Insulin Derivatives

ABSTRACT

The present invention relates to insulin derivatives having a side chain attached either to the -amino group of the N-terminal amino acid residue of the B chain or to the amino group of a Lys residue present in the B chain of the parent insulin via an amide bond which side chain comprises at least one aromatic group; at least one free carboxylic acid group or a group which is negatively charged at neutral pH, a fatty acid moiety with 4 to 22 carbon atoms in the carbon chain; and possible linkers which link the individual components in the side chain together via amide bonds.

FIELD OF THE INVENTION

The present invention relates to novel human insulin derivatives whichare soluble at physiological pH values and have a prolonged profile ofaction. The invention also relates to methods of providing suchderivatives, to pharmaceutical compositions containing them, to a methodof treating diabetes and hyperglycaemia using the insulin derivatives ofthe invention and to the use of such insulin derivatives in thetreatment of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2diabetes, relies to an increasing extent on the so-called intensiveinsulin treatment. According to this regimen, the patients are treatedwith multiple daily insulin injections comprising one or two dailyinjections of long acting insulin to cover the basal insulin requirementsupplemented by bolus injections of a rapid acting insulin to cover theinsulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, onemain type of long acting insulin compositions comprises injectableaqueous suspensions of insulin crystals or amorphous insulin. In thesecompositions, the insulin compounds utilized typically are protamineinsulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions.Thus, in order to secure an accurate dosing, the insulin particles mustbe suspended homogeneously by gentle shaking before a defined volume ofthe suspension is withdrawn from a vial or expelled from a cartridge.Also, for the storage of insulin suspensions, the temperature must bekept within more narrow limits than for insulin solutions in order toavoid lump formation or coagulation.

Another type of long acting insulin compositions are solutions having apH value below physiological pH from which the insulin will precipitatebecause of the rise in the pH value when the solution is injected. Adrawback with these solutions is that the particle size distribution ofthe precipitate formed in the tissue on injection, and thus the releaseprofile of the medication, depends on the blood flow at the injectionsite and other parameters in a somewhat unpredictable manner. A furtherdrawback is that the solid particles of the insulin may act as a localirritant causing inflammation of the tissue at the site of injection.

Human insulin has three primary amino groups: the N-terminal group ofthe A-chain and of the B-chain and the ε-amino group of LysB29. Severalinsulin derivatives which are substituted in one or more of these groupsare known in the prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly)relates to N-carboxyaroyl insulins in which one, two or three primaryamino groups of the insulin molecule has a carboxyaroyl group.

GB Patent No. 1.492.997 (Nat. Res. Dev. Corp.) discloses insulin with acarbamyl substitution at NεB29 with an alleged improved profile ofhypoglycemic effect.

JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.)discloses insulin wherein a fatty acid is bound to the amino group ofPheB1 or to the ε-amino group of LysB29 or to both of these. The statedpurpose of the derivatisation is to obtain a pharmacologicallyacceptable, stable insulin preparation.

Insulins, which in the B30 position have an amino acid having at leastfive carbon atoms which cannot necessarily be coded for by a triplet ofnucleotides, are described in JP laid-open patent application No.57-067548 (Shionogi). The insulin analogues are claimed to be useful inthe treatment of diabetes mellitus, particularly in patients who areinsulin resistant due to generation of bovine or porcine insulinantibodies.

WO 95/07931 (Novo Nordisk A/S) discloses human insulin derivativeswherein the ε-amino group of LysB29 has a lipophilic substituent. Theseinsulin derivatives have a prolonged profile of action and are solubleat physiological pH values.

GP 894095 discloses insulin derivatives wherein the N-terminal group ofthe B-chain and/or the ε-amino group of Lys in position B28, B29 or B30has a substituent of the formula —CO—W—COOH where W can be a long chainhydrocarbon group. These insulin derivatives have a prolonged profile ofaction and are soluble at physiological pH values.

However, there is still a need for insulins having a more prolongedprofile of action than the insulin derivatives known up till now andwhich at the same time are soluble at physiological pH values and have apotency which is comparable to that of human insulin.

SUMMARY OF THE INVENTION

The present invention is based on the recognition that the overallhydrophobicity of an insulin derivative molecule plays an important rolefor the in vivo potency of the derivative.

In one aspect the invention is related to insulin derivatives having aside chain attached either to the α-amino group of the N-terminal aminoacid residue of the B chain or to an ε-amino group of a Lys residuepresent in the A or the B chain of the parent insulin moiety via anamide bond, which side chain comprises at least one aromatic group; atleast one free carboxylic acid group or a group which is negativelycharged at neutral pH, a fatty acid moiety with from 4 to 22 carbonatoms in the carbon chain; and possible one or more linkers linking theindividual components in the side chain together via amide bonds,provided that the fatty acid moiety is not a divalent hydrocarbon chainof the formula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w are integersor one of them is zero so that the sum of v₄ and w₁ is in the range of 6to 30.

In one aspect the aromatic group is arylene or heteroarylene group whichmay be substituted with one or two groups selected from —COOH, —SO₃H,—PO₃H₂ and tetrazolyl.

In another aspect the aromatic group is a 5 to 7 membered heterocyclicring system containing one or more heteroatoms selected from nitrogen,oxygen and sulphur.

In another aspect the aromatic group is 8 to 14 membered bi- ortricyclic heterocyclic ring system containing one or more heteroatomsselected from nitrogen, oxygen and sulphur.

In a further aspect the linker comprises 1-4 amino acid residues linkedtogether via amide bonds of which at least one has a free carboxylicacid group or a group which is negatively charged at neutral pH.

In a further aspect the fatty acid moiety will have from 10-20, from12-18 or from 14 to 18 carbon atoms.

In one aspect the insulin derivative according to the invention has theformula

wherein Ins is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofan Lys residue present in the B chain of the insulin moiety is bound tothe CO— group in the side chain via an amide bond;X₁ is

-   -   —(CH₂)_(n) where n is 1, 2, 3, 4, 5 or 6;    -   NR, where R is hydrogen or —(CH₂)_(p)—COOH; —(CH₂)_(p)—SO₃H;        —(CH₂)_(p)—PO₃H₂; —(CH₂)_(p)—O—SO₃H₂; —(CH₂)_(p)—O—PO₃H₂;        arylene substituted with 1 or 2-(CH₂)_(p)—O—COOH groups;        —(CH₂)_(p)-tetrazolyl, where p is an integer in the range of 1        to 6;    -   —(CR₁R₂)_(q)—NR—CO—, where R₁ and R₂ independently of each other        and independently for each value of q can be H, —COOH, or OH, q        is 1-6 and R is defined as above;    -   —((CR₃R₄)_(q1)—NR—CO—)₂₋₄, where R₃ and R₂ independently of each        other and independently for each value of q₁ can be H, —COOH, or        OH, q₁ is 2-4 and R is defined as above; or    -   a bond        W is arylene or heteroarylene, which may be substituted with one        or two groups selected from the group consisting of —COOH,        —SO₃H, and —PO₃H₂ and tetrazolyl, or W is a bond;        m is 0, 1, 2, 3, 4, 5 or 6;        X is        where R is defined as above; or    -   a bond;        Y is    -   —(CR₁R₂)_(q)—NR—CO—, where R₁ and R₂ independently of each other        and independently for each value of q can be H, —COOH, a bond or        OH, and q is 1-6; and R is defined as above;    -   NR where R is defined as above;    -   —((CR₃R₄)_(q1)—NR—CO)₂₋₄—, where R₃ and R₂ independently of each        other and independently for each value of q₁ can be H, —COOH, or        OH, q₁ is 1-6 and R is defined as above; or    -   a bond;        Q is    -   —(CH₂)_(r)— where r is an integer from 4 to 22;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 4 to 22; or    -   a divalent hydrocarbon chain of the formula        —(CH₂)_(s)-Q₁-(C₆H₄)_(v1)-Q₂-(CH₂)_(W)-Q₃-(C₆H₄)_(v2)-Q₄-(CH₂)_(t)-Q₅-(C₆H₄)_(v3)-Q₆-(CH₂)_(z)—        wherein Q₁-Q₆ independently of each other can be O; S or a bond;        s, w, t and z independently of each other are zero or an integer        from 1 to 10 so that the sum of s, w, t and z is in the range        from 4 to 22, and v₁, v₂, and v₃ independently of each other can        be zero or 1, provided that when W is a bond then Q is not a        divalent hydrocarbon chain of the formula        —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v₄ and w₁ are integers or one        of them is zero so that the sum of v₄ and w₁ is in the range of        6 to 22; and        Z is:    -   —COOH;    -   —CO-Asp;    -   —CO-Glu;    -   —CO-Gly;    -   —CO-Sar;    -   —CH(COOH)₂;    -   —N(CH₂COOH)₂;    -   —SO₃H    -   —PO₃H₂;    -   O—SO₃H;    -   O—PO₃H₂;    -   -tetrazolyl or    -   —O—W₁,    -   where W₁ is arylene or heteroarylene substituted with one or two        groups selected from —COOH, —SO₃H, and —PO₃H₂ and tetrazolyl;    -   provided that if W is a bond and v₁, v₂ and v₃ are all zero and        Q₁₋₆ are all bonds, then Z is —O—W₁        and any Zn²⁺ complex thereof.

In one aspect of the invention, the side is attached to the α-aminogroup of the N-terminal amino acid residue of the B chain of the parentinsulin.

In another aspect of the invention, the side chain is attached to theε-amino group of a Lys residue present in the B chain of the parentinsulin. In a further aspect, the side chain is attached to the ε-aminogroup of a Lys residue present in position 28 or 29 of the B chain.

In one aspect W is phenylene. If the phenylene group comprisessubstituents such substituents can be attached in the 1,4; the 1,3 orthe 1,2 positions.

In a further aspect W is isophtatalic acid or a derivatives thereof.

In another aspect W is 5-7 membered heterocyclic ring system comprisingnitrogen, oxygen or sulphur or a 5 membered heterocyclic ring systemcomprising at least one oxygen such as furan.

Non limiting examples of heterocyclic ring systems are furylene,thienylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene,isoxazolylene, isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene,pyranylene, pyridylene, pyridazinylene, and pyrimidinylene.

In one aspect n is 1 or 2. In another aspect q is 1, 2 or 3. In afurther aspect m is 1 or 2.

In one aspect Q is —(CH₂)_(r)— where r is an integer from 4 to 22, 8 to20, 12 to 20, 12-16, 10-16; 10-20, 14-18 or 14-16.

In another aspect Q is a divalent hydrocarbon chain comprising 1, 2 or 3—CH═CH— groups and a number of —CH₂— groups sufficient to give a totalnumber of carbon atoms in the chain in the range of 4 to 22, 8 to 20, 12to 20, 12-16, 10-16; 10-20, 14-18 or 14-16.

The fatty diacid will typically comprise from 4 to 18, from 6 to 18,from 8 to 16, from 8 to 22, from 8 to 17, from 8 to 15, from 10 to 18,from 10 to 16 and from 6 to 17 carbon atoms in the carbon chain.

Non limiting examples of the fatty diacid moiety are diacids with theformula HOOC—(CH₂)_(r1)—COOH, where r₁ is from 4 to 22

In one aspect Q₁-Q₆ are all a bond and the sum of s, w, t and z is from6 to 18.

In another aspect Q₁-Q₆ are all a bond, the sum of s and z is from 6 to18, and w and t are zero.

In another aspect Q₁-Q₆ are all a bond, v₁ is zero, the sum of s and zis from 6 to 18, and w and t are zero.

In another aspect two of Q₁-Q₆ are oxygen and the other Q's are a bonds.

In one aspect Q₁, Q₂, Q₅ and -Q₆ are all a bond, v₂ is 1 and v₁ and v₃are zero.

In another aspect Q₁, Q₂, Q₅ and -Q₆ are all a bond, v₂ is 1 and v₁ andv₃ are zero and Q₃ and Q₄ are oxygen.

In one aspect R is hydrogen or —(CH)_(p) where p is 1-3.

In one aspect X₁ is —(CH₂)₁₋₄—NH—CO—.

In another aspect X₁ and Y are a bond and X is

where R is —(CH₂)_(p)—COOH, where p is 1-4 or 1-2.

In another aspect X is a bond or α- or γ-Glu.

In one aspect Z is —COOH

In another aspect Z is —CO-Asp.

In another aspect Z is —CO-Glu.

In another aspect Z is —CO-Gly.

In another aspect Z is —CO-Sar.

In another aspect Z is —CH(COOH)₂.

In another aspect Z is —N(CH₂COOH)₂.

In another aspect Z is —SO₃H.

In another aspect Z is —PO₃H.

In another aspect Z is O—SO₃H;

In another aspect Z is O—PO₃H₂;

In another aspect Z is tetrazolyl.

In another aspect Z is —OC₆H₄COOH.

DETAILED DESCRIPTION OF THE INVENTION

The present insulin derivatives are characterized by having a side chainattached to a Lys group in either the B or the A chain or to theN-terminal amino group in the B-chain of the parent insulin moleculewhich side chain comprises an aromatic group and a fatty diacid moiety.

The insulin derivative according to the invention is furthermorecharacterized in having at least one free carboxylic acid group in theside chain and may comprise up to 2 or three free carboxylic acid groupor a group which is negatively charged at neutral pH.

The insulin derivatives will only contain one lysine residue. Thislysine residue may either be in position B29 as in human insulin or inone of position B3, B30 or B23 to B28.

The insulin moiety—in the present text also referred to as the parentinsulin—of an insulin derivative according to the invention can be anaturally occurring insulin such as human insulin or porcine insulin.Alternatively, the parent insulin can be an insulin analogue.

In one group of parent insulin analogues, the amino acid residue atposition A21 is Asn.

In another group of parent insulin analogues, the amino acid residue atposition A21 is Gly. Specific examples from this group of analogues areGly^(A21) human insulin, Gly^(A21) des(B30) human insulin; andGly^(A21)Arg^(B31)Arg^(B32) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B1 has been deleted. A specific example from this group ofparent insulin analogues is desB1 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B30 has been deleted. A specific example from this group ofparent insulin analogues is desB30 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Asp. A specific example from this group of parentinsulin analogues is Asp^(B28) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Lys and the amino acid residue at position B29 is Pro. Aspecific example from this group of parent insulin analogues isLys^(B28)Pro^(B29) human insulin.

In another group of parent insulin analogues the amino acid residue inposition B30 is Lys and the amino acid residue in position B29 is anycodable amino acid except Cys, Met, Arg and Lys. An example is aninsulin analogue where the amino acid residue at position B29 is Thr andthe amino acid residue at position B30 is Lys. A specific example fromthis group of parent insulin analogues is Thr^(B29)Lys^(B30) humaninsulin.

In another group of parent insulin analogues, the amino acid residue atposition B3 is Lys and the amino acid residue at position B29 is Glu. Aspecific example from this group of parent insulin analogues isLys^(B3)Glu^(B29) human insulin.

Examples of insulin derivatives according to the invention are thefollowing compounds:

-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-para C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₃CO)—N-(carboxyethyl)-CH₂-para C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₅CO)—N-(carboxyethyl)-CH₂-para C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-CH₂-para C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-para C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-(-2,5-furanylene)CO]    desB30 human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-meta-C₆H₄CO] desB30    human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-ortho C₆H₄CO]    desB30 human insulin;-   N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—NH—CH₂-para-C₆H₄CO-gamma-Glu] desB30    human insulin;-   N^(εB29)-[5-N—(HOOC(CH₂)₁₄CO)NH-(3-COOH—C₆H₃CO)] desB30 human    insulin;-   N^(εB29)-[5-N—(HOOC(CH₂)₁₆CO)NH-(3-COOH—C₆H₃CO)] desB30 human    insulin;-   N^(εB29)-[5-N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl-Gly)-N-(3-COOH—C₆H₃CO)]    desB30 human insulin and-   N^(εB29ε)-[3-Carboxy-5-(octadecandioyl-N-carboxyethyl-Gly)amino-benzoyl]    desB30 human insulin.

Insulin derivatives according to the invention may be provided in theform of essentially zinc free compounds or in the form of zinccomplexes. When zinc complexes of an insulin derivative according to theinvention are provided, two Zn²⁺ ions, three Zn²⁺ ions or four Zn²⁺ ionscan be bound to each insulin hexamer. Solutions of zinc complexes of theinsulin derivatives will contain mixtures of such species.

In a further aspect of the invention, a pharmaceutical compositioncomprising a therapeutically effective amount of an insulin derivativeaccording to the invention together with a pharmaceutically acceptablecarrier can be provided for the treatment of type 1 diabetes, type 2diabetes and other states that cause hyperglycaemia in patients in needof such a treatment. An insulin derivative according to the inventioncan be used for the manufacture of a pharmaceutical composition for usein the treatment of type 1 diabetes, type 2 diabetes and other statesthat cause hyperglycaemia.

In a further aspect of the invention, there is provided a pharmaceuticalcomposition for treating type 1 diabetes, type 2 diabetes and otherstates that cause hyperglycaemia in a patient in need of such atreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together withpharmaceutically acceptable carriers and additives.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention together with apharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention in mixture with an insulinor an insulin analogue which has a rapid onset of action, together witha pharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In a further aspect of the invention, there is provided a use of aninsulin derivative according to the invention for the manufacture of amedicament for blood glucose lowering.

In a further aspect of the invention, there is provided a use of aninsulin derivative according to the invention for the manufacture of amedicament for treatment of diabetes.

In a further aspect, the present invention relates to insulinderivatives which have an overall hydrophobicity which is essentiallysimilar to that of human insulin.

In one aspect, the insulin derivatives of the present invention have ahydrophobic index, k'rel, which is in the range from about 0.02 to about10, from about 0.1 to about 5; from about 0.5 to about 5; or from about0.5 to about 2.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at physiological pH values.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at pH values in the interval from about 6.5 to about 8.5.

In another aspect, the invention relates to a pharmaceutical compositionwith a prolonged profile of action which comprises an insulin derivativeaccording to the invention.

In another aspect, the invention relates to a pharmaceutical compositionwhich is a solution containing from about 120 nmol/ml to about 2400nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an insulinderivative according to the invention or of a mixture of the insulinderivative according to the invention with a rapid acting insulinanalogue.

The starting product for the acylation, the parent insulin or insulinanalogue or a precursor thereof can be produced by either well-knoworganic synthesis or by well known recombinant production in suitabletransformed microorganisms. Thus the insulin starting product can beproduced by a method which comprises culturing a host cell containing aDNA sequence encoding the polypeptide and capable of expressing thepolypeptide in a suitable nutrient medium under conditions permittingthe expression of the peptide, after which the resulting peptide isrecovered from the culture. As an example desB(30) human insulin can beproduced from a human insulin precursor B(1-29)-Ala-Ala-Lys-A(1-21)which is produced in yeast as disclosed in U.S. Pat. No. 4,916,212. Thisinsulin precursor can then be converted into desB30 human insulin by ALPcleavage of the Ala-Ala-Lys peptide chain to give desB30 human insulinwhich can then be acylated to give the present insulin derivatives.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the parent insulin may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thepolypeptide by hybridisation using synthetic oligonucleotide probes inaccordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the polypeptide may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the peptide is operably linked to additional segments requiredfor transcription of the DNA, such as a promoter. The promoter may beany DNA sequence which shows transcriptional activity in the host cellof choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding the parentinsulin in a variety of host cells are well known in the art, cf. forinstance Sambrook et al., supra.

The DNA sequence encoding the parent insulin may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the parentinsulin, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook, J, Fritsch, E Fand Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1989).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the parentinsulin and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

The parent insulin molecule is then converted into the insulinderivatives of the invention by introducing of the relevant side chainin either the B1 position or in the chosen Lys position in the B-chain.The side chain can be introduced by any convenient method and manymethods are disclosed in the prior art for acylation of an amino group.More details will appear from the following examples.

Pharmaceutical Compositions

Pharmaceutical compositions containing an insulin derivative accordingto the present invention may be administered parenterally to patients inneed of such a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. Furtheroptions are to administer the insulin nasally or pulmonally, preferablyin compositions, powders or liquids, specifically designed for thepurpose.

Injectable compositions of the insulin derivatives of the invention canbe prepared using the conventional techniques of the pharmaceuticalindustry which involve dissolving and mixing the ingredients asappropriate to give the desired end product. Thus, according to oneprocedure, an insulin derivative according to the invention is dissolvedin an amount of water which is somewhat less than the final volume ofthe composition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

In a further aspect of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeaspect of the invention.

In a further aspect of the invention the formulation further comprises apharmaceutically acceptable preservative which may be selected from thegroup consisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further aspect of the invention the preservative ispresent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furtheraspect of the invention the preservative is present in a concentrationfrom 0.1 mg/ml to 5 mg/ml. In a further aspect of the invention thepreservative is present in a concentration from 5 mg/ml to 10 mg/ml. Ina further aspect of the invention the preservative is present in aconcentration from 10 mg/ml to 20 mg/ml. Each one of these specificpreservatives constitutes an alternative aspect of the invention. Theuse of a preservative in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

In a further aspect of the invention the formulation further comprisesan isotonic agent which may be selected from the group consisting of asalt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid(e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one aspect the sugar additive is sucrose. Sugar alcohol is defined asa C₄-C₈ hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one aspect the sugar alcohol additive is mannitol. Thesugars or sugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to the amount used, as long as thesugar or sugar alcohol is soluble in the liquid preparation and does notadversely effect the stabilizing effects achieved using the methods ofthe invention. In one aspect, the sugar or sugar alcohol concentrationis between about 1 mg/ml and about 150 mg/ml. In a further aspect of theinvention the isotonic agent is present in a concentration from 1 mg/mlto 50 mg/ml. In a further aspect of the invention the isotonic agent ispresent in a concentration from 1 mg/ml to 7 mg/ml. In a further aspectof the invention the isotonic agent is present in a concentration from 8mg/ml to 24 mg/ml. In a further aspect of the invention the isotonicagent is present in a concentration from 25 mg/ml to 50 mg/ml. Each oneof these specific isotonic agents constitutes an alternative aspect ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoneand glycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of an insulin derivativeaccording to the present invention may, for example, be prepared asdescribed in European Patent No. 272097 (to Novo Nordisk A/S).

Compositions containing insulins of this invention can be used in thetreatment of states which are sensitive to insulin. Thus, they can beused in the treatment of type 1 diabetes, type 2 diabetes andhyperglycaemia for example as sometimes seen in seriously injuredpersons and persons who have undergone major surgery. The optimal doselevel for any patient will depend on a variety of factors including theefficacy of the specific insulin derivative employed, the age, bodyweight, physical activity, and diet of the patient, on a possiblecombination with other drugs, and on the severity of the state to betreated. It is recommended that the daily dosage of the insulinderivative of this invention be determined for each individual patientby those skilled in the art in a similar way as for known insulincompositions.

Where expedient, the insulin derivatives of this invention may be usedin mixture with other types of insulin, e.g. insulin analogues with amore rapid onset of action. Examples of such insulin analogues aredescribed e.g. in the European patent applications having thepublication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo NordiskA/S) and EP 383472 (Eli Lilly & Co.).

In a further aspect of the present invention the present compounds areadministered in combination with one or more further active substancesin any suitable ratios. Such further active agents may be selected fromantidiabetic agents, antihyperlipidemic agents, antiobesity agents,antihypertensive agents and agents for the treatment of complicationsresulting from or associated with diabetes.

Suitable antidiabetic agents include insulin, GLP-1 (glucagon likepeptide-1) derivatives such as those disclosed in WO 98/08871 (NovoNordisk A/S), which is incorporated herein by reference, as well asorally active hypoglycemic agents.

Suitable orally active hypoglycemic agents preferably includeimidazolines, sulfonylureas, biguanides, meglitinides,oxadiazolidinediones, thiazolidinediones, insulin sensitizers,α-glucosidase inhibitors, agents acting on the ATP-dependent potassiumchannel of the pancreatic β-cells eg potassium channel openers such asthose disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (NovoNordisk A/S) which are incorporated herein by reference, potassiumchannel openers, such as ormitiglinide, potassium channel blockers suchas nateglinide or BTS-67582, glucagon antagonists such as thosedisclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and AgouronPharmaceuticals, Inc.), all of which are incorporated herein byreference, GLP-1 agonists such as those disclosed in WO 00/42026 (NovoNordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporatedherein by reference, DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase(protein tyrosine phosphatase) inhibitors, inhibitors of hepatic enzymesinvolved in stimulation of gluconeogenesis and/or glycogenolysis,glucose uptake modulators, GSK-3 (glycogen synthase kinase-3)inhibitors, compounds modifying the lipid metabolism such asantihyperlipidemic agents and antilipidemic agents, compounds loweringfood intake, and PPAR (peroxisome proliferator-activated receptor) andRXR (retinoid X receptor) agonists such as ALRT-268, LG-1268 or LG-1069.

DEFINITIONS

With “desB30” or “B(1-29)” is meant a natural insulin B chain or ananalogue thereof lacking the B30 amino acid residue and “A(1-21)” meansthe natural insulin A chain or an analogue thereof. The C-peptide andits amino acid sequence are indicated in the three letter amino acidcode. DesB30,desB29 human insulin is a human insulin lacking B29 andB30.

With “B1”, “A1” etc. is meant the amino acid residue in position 1 inthe B chain of insulin (counted from the N-terminal end) and the aminoacid residue in position 1 in the A chain of insulin (counted from theN-terminal end), respectively. The amino acid residue in a specificposition may also be denoted as e.g. Phe^(B1) which means that the aminoacid residue in position B1 is a phenylalanine residue.

With “Insulin” as used herein is meant human insulin with disulfidebridges between Cys^(A7) and Cys^(B7) and between Cys^(A20) andCys^(B19) and an internal disulfide bridge between Cys^(A6) andCys^(A11), porcine insulin and bovine insulin.

By “insulin analogue” as used herein is meant a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, for example that of human insulin, bydeleting and/or substituting at least one amino acid residue occurringin the natural insulin and/or by adding at least one amino acid residue.The added and/or substituted amino acid residues can either be codableamino acid residues or other naturally occurring amino acid residues orpurely synthetic amino acid residues.

The insulin analogues may be such wherein position 28 of the B chain maybe modified from the natural Pro residue to Asp, Lys, or Ile. Lys inposition B29 may also be modified to Pro. Furthermore B30 may be Lys inwhich case B29 is different from Cys, Met, Arg and Lys.

Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His,Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala,Ser, or Thr and in particular to Gly. Furthermore, Asn at position B3may be modified to Lys or Asp. Further examples of insulin analogues aredes(B30) human insulin, insulin analogues wherein one or both of B1 andB2 have been deleted; insulin analogues wherein the A-chain and/or theB-chain have an N-terminal extension and insulin analogues wherein theA-chain and/or the B-chain have a C-terminal extension. Further insulinanalogues are such wherein one or more of B26-B30 have been deleted.

By “insulin derivative” as used herein is meant a naturally occurringinsulin or an insulin analogue which has been chemically modified, e.g.by introducing a side chain in one or more positions of the insulinbackbone or by oxidizing or reducing groups of the amino acid residuesin the insulin or by acylating a free amino group or a hydroxy group.

The expression “a codable amino acid” or “a codable amino acid residue”is used to indicate an amino acid or amino acid residue which can becoded for by a triplet (“codon”) of nucleotides.

hGlu is homoglutamic acid.

α-Asp is the L-form of —HNCH(CO—)CH₂COOH.

β-Asp is the L-form of —HNCH(COOH)CH₂CO—.

α-Glu is the L-form of —HNCH(CO—)CH₂CH₂COOH.

γ-Glu is the L-form of —HNCH(COOH)CH₂CH₂CO—.

α-hGlu is the L-form of —HNCH(CO—)CH₂CH₂CH₂COOH.

δ-hGlu is the L-form of —HNCH(COOH)CH₂CH₂CH₂CO—.

β-Ala is —NH—CH₂—CH₂—CO—.

Sar is sarcosine (N-methylglycine).

The expression “an amino acid residue having a carboxylic acid group inthe side chain” designates amino acid residues like Asp, Glu and hGlu.The amino acids can be in either the L- or D-configuration. If nothingis specified it is understood that the amino acid residue is in the Lconfiguration.

The expression “an amino acid residue having a neutral side chain”designates amino acid residues like Gly, Ala, Val, Leu, Ile, Phe, Pro,Ser, Thr, Cys, Met, Tyr, Asn and Gln.

With “activated acid” is meant a carboxylic acid in which an activatedleaving group has been attached to the acyl carbon enabling reactionwith an amino group under formation of an amide bond and release of theleaving group. Activated fatty acids may be activated esters of fattyacids, activated amides of fatty acids and anhydrides or chlorides.Activated fatty acid includes derivatives thereof such asN-hydroxybenzotriazole and N-hydroxysuccinimide.

With “fatty acid” is meant a linear or branched carboxylic acids havingat least 2 carbon atoms and being saturated or unsaturated. Examples offatty acids are capric acid, lauric acid, tetradecanoic acid (myristicacid), pentadecanoic acid, palmitic acid, heptadecanoic acid, andstearic acid.

The term “arylene” as used herein is intended to include divalent,carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9to 14 membered bi- and tricyclic, divalent, carbocyclic, aromatic ringsystems. Representative examples are phenylene, biphenylylene,naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene,azulenylene and the like. Arylene is also intended to include thepartially hydrogenated derivatives of the ring systems enumerated above.Non-limiting examples of such partially hydrogenated derivatives are1,2,3,4-tetrahydronaphthylene, 1,4-dihydronaphthylene and the like.

The term “heteroarylene” as used herein is intended to include divalent,aromatic, heterocyclic ring systems containing one or more heteroatomsselected from nitrogen, oxygen and sulfur such as 5 to 7 memberedmonocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclicring systems containing one or more heteroatoms selected from nitrogen,oxygen and sulfur. Representative examples are furylene, thienylene,pyrrolylene, oxazolylene, thiazolylene, imidazolylene, isoxazolylene,isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene, pyranylene,pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene,1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene,1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene,1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene,1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene,thiadiazinylene, indolylene, isoindolylene, benzofurylene,benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene,benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene,quinazolinylene, quinolizinylene, quinolinylene, isoquinolinylene,quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene,azepinylene, diazepinylene, acridinylene and the like. Heteroaryl isalso intended to include the partially hydrogenated derivatives of thering systems enumerated above. Non-limiting examples of such partiallyhydrogenated derivatives are 2,3-dihydrobenzofuranylene, pyrrolinylene,pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene,oxazepinylene and the like.

The term “optionally substituted” as used herein means that the groupsin question are either unsubstituted or substituted with one or more ofthe substituents specified. When the groups in question are substitutedwith more than one substituent the substituents may be the same ordifferent.

When an insulin derivative according to the invention is stated to be“soluble at physiological pH values” it means that the insulinderivative can be used for preparing insulin compositions that are fullydissolved at physiological pH values. Such favourable solubility mayeither be due to the inherent properties of the insulin derivative aloneor a result of a favourable interaction between the insulin derivativeand one or more ingredients contained in the vehicle.

The following abbreviations and methods have been used in thespecification and examples:

Bzl=Bn: benzyl

DIPEA=DIEA: N,N-diisopropylethylamine

DMF: N,N-dimethylformamide

tBu: tert-butyl

TSTU: O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

THF: Tetrahydrofuran

EtOAc=AcOEt: Ethyl acetate

DIPEA: Diisopropylethylamine

HOAt: 1-Hydroxy-7-azabenzotriazole

TEA: triethyl amine

Su: succinimidyl=2,5-dioxo-pyrrolidin-1-yl

TFA: trifluoracetic acid

DCM: dichloromethane

DMSO: dimethyl sulphoxide

HOBt: 1-hydroxybenzotriazole

TRIS: Triisopropylsilane

EDAC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

NMP: 1-methyl-2-pyrrolidone

TLC: Thin Layer Chromatography

RT: room temperature

R_(t): Retention time

MeOH: methanol

DCC: Dicyclohexylcarondiimide

AcOH: Acetic acid

DIC: Diisopropylcarbodiimide

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

EXAMPLES

HPLC-MS: The following instrumentation was used:

Hewlett Packard series 1100 G1312A Bin Pump

Hewlett Packard series 1100 Column compartment

Hewlett Packard series 1100 G1315A DAD diode array detector

Hewlett Packard series 1100 MSD

Sedere 75 Evaporative Light Scattering detector

The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs containing:

A: 0.01% TFA in water

B: 0.01% TFA in acetonitrile

The analysis is performed at 40 C by injecting an appropriate volume ofthe sample (preferably 1 μl) onto the column which is eluted with agradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following:

Column: Waters Xterra MS C-18×3 mm id 5 μm

Gradient: 5%-100% acetonitrile linear during 7.5 min at 1.5 ml/min

Detection: 210 nm (analogue output from DAD)

-   -   ELS (analogue output from ELS)

After the DAD the flow was divided yielding approx 1 ml/min to the ELSand 0.5 ml/min to the MS.

HPLC-MS (method fast grad): The following instrumentation was used:

Hewlett Packard series 1100 G1312A Bin Pump

Hewlett Packard series 1100 Column compartment

Hewlett Packard series 1100 G1315A DAD diode array detector

Hewlett Packard series 1100 MSD Sedere 75 Evaporative Light Scatteringdetector

The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs containing:

A: 0.05% TFA in water

B: 0.05% TFA in acetonitrile

The analysis is performed at 40 C by injecting an appropriate volume ofthe sample (preferably 1 μl) onto the column which is eluted with agradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following:

Column: Waters Xterra MS C-18×3 mm id 5 μm

Gradient: 5%-95% acetonitrile linear during 3 min at 2.7 ml/min

Detection: 210 nm (analogue output from DAD)

-   -   ELS (analogue output from ELS)

After the DAD the flow was divided yielding approx 1 ml/min to the ELSand 0.5 ml/min to the MS.

HPLC-MS (50-99)

The same instruments and procedure as in the fast grad method is used.The only difference is that the gradient runs from 50-99% acetonitrile.

HPLC (Neutral)

-   -   Buffer A: 10 mMtris, 15 mM (NH₄)₂SO₄, pH adjusted to 7.3 with 4N        H₂SO₄, 20% v/v acetonitrile    -   Buffer B: 80% v/v acetonitrile    -   Flow: 1.5 ml/min    -   Gradient: 0-20 min 10-50% B    -   Column: Phenomerex, Jupiter 4.6 mm×150 mm, C₄, 5μ, 300 Å    -   Column temperature: 40° C.

HPLC-MS (Sciex)

The following instrumentation is used:

-   -   Hewlett Packard series 1100 G1312A Bin Pump    -   Hewlett Packard series 1100 G1315A DAD diode array detector    -   Sciex3000 triplequadropole mass spectrometer    -   Gilson 215 micro injector    -   Sedex55 evaporative light scattering detector

Pumps and detectors are controlled by MassChrom 1.1.1 software runningon a Macintosh G3 computer. Gilson Unipoint Version 1.90 controls theauto-injector.

The HPLC pump is connected to two eluent reservoirs containing:

A: 0.01% TFA in water

B: 0.01% TFA in acetonitrile

The analysis is performed at room temperature by injecting anappropriate volume of the sample (preferably 10 μl) onto the column,which is eluted, with a gradient of acetonitrile. The eluate from thecolumn passed through the UV detector to meet a flow splitter, whichpassed approximately 30 μl/min (1/50) through to the API Turbo ion-sprayinterface of API 3000 spectrometer. The remaining 1.48 ml/min (49/50) ispassed through to the ELS detector.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table. Column Waters X-Terra C18, 5μ,50 mm × 3 mm id Gradient 5%-90% acetonitrile linearly during 7.5 min at1.5 ml/min Detection 210 nm (analogue output from DAD) MS ionisationmode API Turbo ion-spray ELS Gain 8 and 40° C.

Example 1 General Procedure A, Acylation Using desB30 Human InsulinN^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] des B30human insulin Step 1: Synthesis of4-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid

Tert-butyl 3-aminopropanoate hydrochloride (5 g, 27.7 mmol) wasdissolved in methanol (150 mL). Diisopropylethylamin (4.73 mL, 27.7mmol) was added followed by 4-carboxybenzaldehyde. The mixture is heatedto reflux for 1 hour. After cooling to room temperature sodiumcyanoborohydride (1.77 g, 22.1 mmol) was added under nitrogen andstirred for 1 hour at room temperature. Acetic acid (15 mL) was addedand the mixture was stirred for an additional 1 hour. The mixture waspoured into water (300 mL) and stirred at room temperature over night.The water solution was washed with ethyl acetate (3×250 mL). The organicphase dried (Na₂SO₄) and solvent removed in vacuo to yield the crudeproduct as an oil which solidifies by standing. The crude product wasused in the next step without further purification.

HPLC-MS: m/z=(280); R_(t)=2.09 min.

Step 2: Synthesis of4-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)amino]methyl}benzoicacid

Hexadecanedioic acid mono-tert-butyl ester (0.3 g, 0.88 mmol) wasdissolved in ethyl acetate.N-Ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.167 g,0.88 mmol) and 1-hydroxy-7-azabenzotriazole (0.119 g, 0.88 mmol) wasadded and the mixture was stirred at 50° C. for 1 hour. After cooling toroom temperature, diisopropylethylamin (0.45 mL, 2.63 mmol) was addedfollowed by 4-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid(0.489 g, 1.75 mmol). The mixture was stirred overnight under nitrogenat room temperature. The mixture was separated between ethyl acetate(100 mL) and water (2×50 mL). The organic phase was dried (Na₂SO₄),solvent removed in vacuo. The crude product was purified by RP-HPLC onC18-column, buffer A: 0.1% TFA, buffer B: MeCN+0.1% TFA; gradient80-100% B to yield the title compound (145 mg, 27%).

¹H NMR (Acetone-d₆): δ 8.04 (dd, 2H), 7.40 (dd, 2H), 4.75 (d, 2H), 3.60(q, 2H), 1.55 (m, 4H), 1.45-1.15 (m, 38H).

Step 3: Synthesis of4-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)amino]methyl}benzoicacid 2,5-dioxopyrrolidin-1-yl ester

4-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)amino]methyl}benzoicacid (70 mg, 0.12 mmol) was dissolved in THF (5 mL). The mixture wascooled with an ice bath. Diisopropylethylamin (0.024 mL, 0.14 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (42mg, 0.14 mmol) was added. The mixture was stirred under nitrogen at 0°C. After 30 minutes the ice cooling was removed and the mixture wasstirred for an additional 3 hours. Solvent removed in vacuo flowed byreevaporation from toluene. The crude product was dissolved in ethylacetate (25 mL), washed with water (10 mL). The organic phase dried(Na₂SO₄), solvent removed in vacuo to yield the title compound (73 mg,87%) which was used in subsequent step.

HPLC-MS: m/z=(723 (M+Na)); R_(t)=6.24 min.

Step 4: Synthesis ofB29N(esp)(4-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]-methyl}benzoyl)desB30 human insulin

Human DesB30 insulin (594 mg, 0.104 mmol) was dissolved in aqueousNa₂CO₃ (100 mM, 5 mL).4-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)amino]methyl}benzoicacid 2,5-dioxopyrrolidin-1-yl ester (73 mg, 0.104 mmol) was dissolved inacetonitrile (3 mL) and added. The mixture was stirred very slowly for 1hour at RT. pH was adjusted to 5.5 with aqueous HCl (1N) and thesuspension was allowed to stand for 10 minutes at 0° C. The precipitatewas isolated by centrifugation and treated with TFA/water (95:5, 12 mL)for 30 minutes. Poured into ice cooled diethylether (30 mL), and thecrude product was isolated by centrifuge and purified with RP-HPLC on aWaters Prep LC2000, on C18, 5 cm×20 cm, flow 20 ml/min usingacetonitrile/water 33-53% gradient containing 0.1% TFA. Fraktionscontaining product was collected and lyophilized. To the lyophilizedmaterial was added water (7.2 mL) and pH adjusted to 8.98 with 1 N+0.1 NNaOH. The pH was adjusted back to 5.2-5.5 with 0.1 N HCl. The productprecipitated, isolated by centrifuge and lyophilized to give the titlecompound.

Example 2 General Procedure B. Acylation Using A1,B1-diBoc desB30 HumanInsulin N^(εB29)—[N—(HOOC(CH₂)₁₃CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO]desB30 human insulin Step 1: Synthesis of4-{[(2-tert-Butoxycarbonyl-ethyl)-(14-tert-butoxycarbonyl-tetradecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester

The compound was prepared similar as described in step 2 and step 3 ingeneral procedure A using pentadecanedioic acid mono-tert-butyl esterinstead.

HPLC-MS: m/z=(709 (M+Na)); R_(t)=6.03 min.

Step 2: SynthesisN^(εB29)(4-{[(2-Carboxyethyl)-(14-carboxytetradecanoyl)amino]-methyl}benzoyl)desB30 human Insulin

A1B1 BocBoc desB30 insulin (Kurtzhals P; Havelund S; Jonassen I; KiehrB; Larsen U D; Ribel U; Markussen J Biochemical Journal, 1995, 312,725-731) (0.1 g, 0.017 mmol) was dissolved in DMSO (2 mL). Triethylamin(0.024 mL, 0.17 mmol) was added.4-{[(2-tert-Butoxycarbonyl-ethyl)-(14-tert-butoxycarbonyl-tetradecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester was dissolved in THF (1 mL) andadded. The mixture was shaken at RT for 1 hour. The solution was cooledwith an ice bath, and water (5 mL) was added. The pH was adjusted to 5.2with 1N HCl. The mixture was allowed to precipitate for 1 hour at 5° C.The precipitate was isolated by centrifuge and treated with TFA 10 mLfor 15 minutes. Poured into ice cooled diethylether (35 mL), and thecrude product was isolated by centrifuge and purified on C-18 RP-HPLC 5cm×20 cm, flow 20 ml/min using acetonitrile/water 25-45% gradientcontaining 0.1% TFA. Fractions containing product were collected,lyophilized. The lyophilized material was added water (7.2 mL) and pHadjusted to 8.98 with 1 N+0.1 N NaOH. The pH was adjusted back to5.2-5.5 with 0.1 N HCl. The product was precipitated, isolated bycentrifugation and lyophilized to give the title compound.

HPLC-MS: m/z=1542 (m/4), 1234 (m/5); R_(t)=3.55 min.

Example 3 General Procedure BN^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] des B30human insulin Step 1: Synthesis of4-{[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonylheptadecanoyl)amino]methyl}benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester

The compound was prepared similar as described in step 2 and step 3 ingeneral procedure A using octadecanedioic acid mono-tert-butyl ester instead.

HPLC-MS: m/z=(752 (M+Na)); R_(t)=6.62 min

Step 2: Synthesis ofN^(εB29)-(4-{[(2-Carboxyethyl)-(17-carboxyheptadecanoyl)-amino]methyl}benzoyl)desB30 human insulin

Compound from step 1 was reacted with from A1,B1-diBoc desB30 insulin asdescribed in general procedure B. The work up was similar using agradient 45-70% acetonitrile/water containing 0.1% TFA. The pooledfractions containing product were lyophilized and dissolved in 2.5% NH₃1 mL and diluted to 10 mL and subjected to purification on an ÄKTApurifier employing a reversed phase HPLC, Jupiter 5269, C4 250/20 mm, 15μM, 300 Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM (NH₄)₂SO₄in 20% EtOH, pH 7.3 and a B-buffer 80% EtOH. The product was eluted witha gradient 15-60% B with 8 ml/min. The appropriate fraction were pooledand eluted on a sep pak with 70% CH₃CN containing 0.1% TFA. Precipitatedand lyophilized to yield the desired product.

Maldi: 6199.2

Example 4 General Procedure BN^(εB29)—[N—(HOOC(CH₂)₁₅CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB30human insulin Step 1: Synthesis of heptadecanedioic acid mono-tert-butylester

2-Methoxycarbonyl-heptadecanedioic acid 1-methyl ester

Sodium (1.11 g, 48.2 mmol) was dissolved in dry methanol (30 ml) andheated to 50° C. Dimethyl malonate (5.87 ml, 51.4 mmol) was added over15 min. The mixture was heated to reflux and a suspension of15-bromopentadecanoic acid (5 g, 15.6 mmol) in dry methanol (50 ml) wasadded over 45 min. The resulting mixture was refluxed for another 30min. After cooling to RT, water was added and the mixture wasconcentrated. Water was added to the residue an made alkaline with 1 NNaOH and extracted with ether (1×50 ml). The aqueous layer was acidifiedwith 1N HCl and extracted with ether (3×30 ml). The combined organiclayers was dried (Na₂SO₄) and concentrated to give the title compound in98% (5.7 g) yield.

HPLC-MS: 395 (M+Na), rt 5.38. ¹H-NMR (CDCl₃): δ 1.2-1.35 (m, 22H), 1.65(pent, 2H), 1.90 (m, 2H), 2.34 (t, 2H), 3.37 (t, 1H), 3.71 ppm (s, 6H).

Heptadecanedioic Acid

2-Methoxycarbonyl-heptadecanedioic acid 1-methyl ester (4.63, 12.4 mmolg) was dissolved in 20% aqueous KOH (15 ml) by heating. The resultingsolution was refluxed for 2.5 h. The cold reaction mixture was carefullyconcentrated. The residue was suspended in water (30 ml) on an ice bathand acidified with 10% aqueous HCl. The resulting slurry was refluxedfor 2 h. After cooling the precipitate was isolated by filtration anddried over night in vacuo. The compound was decarboxylated by heatingunder stirring at 140° C. for 2 h. (the reaction should be followed,heating to 1800 might be necessary). The crude product (4.0 g, 100%) wasused without further purification. HPLC-MS: 323 (M+Na), R_(t) 4.61.¹H-NMR (DMSO-d₆): δ 1.22 (br s, 22H) 1.47 (m, 4H), 2.18 (t, 4H).

Heptadecanedioic Acid Mono-Tert-Butyl Ester

The crude heptadecanedioic acid (0.99 g, 3.3 mmol) was dissolved intoluene (15 ml) at 115° C. N,N-dimethylformamide di-tert-butylacetale(0.79 ml, 3.3 mmol) was added dropwise over 10 min. After refluxing for1 h more N,N-dimethylformamide di-tert-butylacetale (0.79 ml) was addedover 10 min. After refluxing for another 1 h, a last eq ofN,N-dimethylformamide di-tert-butylacetale (0.79 ml) was added over 10min. Reflux was continued for 1 h. On cooling to RT a precipitateappeared, this was filtered off (diacid). The mother liqueour wasextracted with water (25 ml) and DCM (25 ml). The organic layer wasdried and concentrated. The residue was purified by flash chromatographyusing DCM/MeOH 15:1 as eluent. Heptadecanedioic acid mono-tert-butylester was isolated in 33% yield (0.330 g). HPLC-MS: 379 (M+Na), rt 6.11.¹H-NMR (DMSO-d₆): δ 1.22 (br s, 22H), 1.39 (s, 9H), 1.47 (m, 4H), 2.16(t, 2H), 2.19 ppm (t, 2H).

Step 2: Synthesis of4-{[(2-tert-Butoxycarbonylethyl)-(16-tert-butoxycarbonylhexadecanoyl)amino]methyl}benzoicacid 2,5-dioxopyrrolidin-1-yl ester

The compound was prepared similar as described in step 2 and step 3 ingeneral procedure A using heptadecanedioic acid mono-tert-butyl ester instead.

Step 3: Synthesis ofN^(εB29)-(4-{[(2-Carboxyethyl)-(16-carboxyhexadecanoyl)amino]-methyl}benzoyl)desB30 human insulin

4-{[(2-tert-Butoxycarbonylethyl)-(16-tert-butoxycarbonylhexadecanoyl)-amino]methyl}benzoicacid 2,5-dioxopyrrolidin-1-yl ester was reacted with A1,B1-diBoc desB30insulin as described in general procedure B. The work up was similarusing a gradient 28-48% acetonitrile/water containing 0.1% TFA.

HPLC-MS: m/z=1549 (m/4), 1239 (m/5); R_(t)=3.53 min.

Example 5 General Procedure BN^(εB29)-[(5-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}furan-2-carbonyl)desB30 human insulin Step 1:5-[(2-tert-Butoxycarbonylethylamino)methyl]furan-2-carboxylic acid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using 5-formylfuran-2-carboxylic acidinstead of 4-carboxybenzaldehyde

¹H NMR (CDCl₃): δ 7.12 (d, 1H), 6.65 (d, 1H), 4.37 (s, 2H), 3.35 (t,2H), 2.80 (t, 2H), 1.45 (s, 9H).

Step 2:5-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)amino]-methyl}furan-2-carboxylicacid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using5-[(2-tert-Butoxycarbonylethylamino)methyl]furan-2-carboxylic acidinstead of 4-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid.

¹H NMR (DMSO-d₆): δ 7.15 (dd, 1H), 6.45 (dd, 1H), 4.57 (d, 2H),2.43-2.10 (m, 6H), 1.60-1.20 (m, 42H).

HPLC-MS: m/z=(616 (M+Na), 538 (lose of tert-butyl, 482 (lose of twotert-butyl), R_(t)=6.17 min.

Step 3:5-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)-amino]methyl}-furan-2-carboxylicacid 2,5-dioxo-pyrrolidin-1-yl ester

This compound was synthesized using a similar procedure as described instep 3 in general procedure A

HPLC-MS: m/z=(713 (M+Na), R_(t)=6.4 min.

Step 4:N^(εB29)-[(5-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}furan-2-carbonyl)desB30 human insulin

5-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)amino]methyl}furan-2-carboxylicacid 2,5-dioxo-pyrrolidin-1-yl ester was reacted with A1,B1-diBoc desB30insulin as described in general procedure B. The work up was similarusing a gradient 25-45% acetonitrile/water containing 0.1% TFA. Thepooled fractions containing product was lyophilized and dissolved in2.5% NH₃ 1 mL and diluted to 38 mL and subjected to purification on anÄKTA purifier employing a reversed phase HPLC, Jupiter 5269, C4 250/20mm, 15 μM, 300 Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM(NH₄)₂SO₄ in 20% EtOH, pH7.3 and a B-buffer 80% EtOH. The product waseluted with a gradient 15-60% B with 8 ml/min. The appropriate fractionswere pooled and eluted on a sep pak with 3 mL 70% CH₃CN containing 0.1%TFA. Precipitated and lyophilized to yield the desired product.

MS. m/z=6169.

Example 6 General Procedure BN^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-meta C₆H₄CO] desB30human insulin Step 1: 3-[(2-tert-Butoxycarbonylethylamino)methyl]benzoicacid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using 3-carboxybenzaldehyde instead of4-carboxybenzaldehyde

HPLC-MS (Method fast grad): m/z=(302, M+Na); R_(t)=1.1 min.

Step 2:3-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)amino]methyl}benzoicacid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using3-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid instead of4-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid.

HPLC-MS (Method fast grad): m/z=(603, M+1); R_(t)=3.09 min.

Step 3:2-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester

This compound was synthesized using a similar procedure as described instep 3 in general procedure A

HPLC-MS (Method fast grad): m/z=723, (M+Na); R_(t)=3.15 min.

Step 4: Synthesis ofN^(εB29)-(3-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]-methyl}benzoyl)desB30 human insulin

3-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester was reacted with A1B1BocBoc desB30insulin as described in general procedure B. The work up was similarusing a gradient 25-45% acetonitrile/water containing 0.1% TFA. Thepooled fractions containing product were precipitated and lyophilized.

HPLC-MS: m/z=1236 (m/5), 1030 (m/6); R_(t)=3.7 min.

Example 7 General Procedure BN^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-ortho C₆H₄CO] desB30human insulin Step 1: 2-[(2-tert-Butoxycarbonylethylamino)methyl]benzoicacid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using 2-carboxybenzaldehyde instead of4-carboxybenzaldehyde

HPLC-MS (Method fast grad): m/z=280, (M+1); R_(t)=1.13 min.

Step 2:3-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)amino]methyl}benzoicacid

This compound was synthesized using a similar procedure as described instep 1 in general procedure A using2-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid instead of4-[(2-tert-Butoxycarbonylethylamino)methyl]benzoic acid.

HPLC-MS (Method fast grad): m/z=604; R_(t)=3.09 min.

Step 3:3-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester

This compound was synthesized using a similar procedure as described instep 3 in general procedure A

HPLC-MS (Method fast grad): m/z=(723, M+Na); R_(t)=3.15 min.

Step 4: Synthesis ofN^(εB29)(3-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]-methyl}benzoyl)desB30 human insulin

2-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-pentadecanoyl)-amino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester was reacted with A1,B1-diBocdes(B30) insulin as described in general procedure B. The work up wassimilar using a gradient 28-48% acetonitrile/water containing 0.1% TFA.The pooled fractions containing product was precipitated and lyophilizedThe pooled fractions containing product was lyophilized and dissolved in2.5% NH₃ 1 mL and diluted to 38 mL and subjected to purification on anÄKTA purifier employing a reversed phase HPLC, Jupiter 5269, C4 250/20mm, 15 μM, 300 Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM(NH₄)₂SO₄ in 10% Acetonitrile, pH7.3 and a B-buffer 70% Acetnotrile. Theproduct was eluted with a gradient 27-33% B with 6 ml/min over 90minutes The appropriate fractions were pooled and eluted on a sep packwith 3 mL 70% CH₃CN containing 0.1% TFA, precipitated and lyophilized toyield the desired product.

Example 8 N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-para-C₆H₄CO]desB30 human insulin Step 1:4-(15-tert-Butoxycarbonylpentadecanoylamino)benzoic acid methyl ester

Hexadecanedioic acid mono-tert-butyl ester (0.4 g, 1.17 mmol) wasdissolved in NMP (6 mL). N-Ethyl-N-(3-dimethylaminopropyl)-carbodiimidehydrochloride (0.223 g, 0.1.17 mmol) and 1-hydroxy-7-azabenzotriazole(0.159 g, 1.17 mmol) was added and the mixture was stirred at 50° C. for1 hour and then allowed to cool to room temperature.Diisopropylethylamin (0.6 mL, 3.5 mmol) was added followed by methyl4-aminobenzoate (0.353 g, 2.34 mmol). The mixture was stirred overnightunder nitrogen at room temperature. The mixture was poured intosaturated aqueous NaCl (50 mL), washed with diethylether (3×100 mLbetween ethyl acetate. The organic phases were collected, dried (Na₂SO₄)and solvent removed in vacuo. The crude material was purified on silicausing ethyl acetate/heptane (50:50), to give pure4-(15-tert-Butoxycarbonylpentadecanoylamino)benzoic acid methyl ester(235 mg, 42%)

¹H-NMR (CDCl₃) δ 8.00 (d, 2H), 7.61 (d, 2H), 7.32 (br s, 1H), 3.90 (s,3H), 2.37 (t, 2H), 2.20 (t, 2H), 1.70 (m, 2H), 1.43 (s, 9H), 1.40-1.20(m, 20H)

Step 2:4-[tert-Butoxycarbonylmethyl-(15-tert-butoxycarbonylpentadecanoyl)amino]benzoicacid

4-(15-tert-Butoxycarbonylpentadecanoylamino)benzoic acid methyl ester(235 mg, 0.495 mmol) was evaporated twice from dry pyridine and oncefrom dry acetonitrile, dissolved in dry DMF (4 mL) under nitrogen. 60%NaH (14 mg, 0.594 mmol) was added and the mixture was stirred for 20minutes at room temperature under nitrogen, tert-Butyl bromoacetate(0.11 mL, 0.742 mmol) was added and the mixture was stirred for 1 hour,the reaction was quenched with ice and separated between water (50 mL)and diethylether (75 mL). The organic phase was dried (Na₂SO₄) andsolvent remove in vacuo. The crude material was dissolved in ethanol (4mL). NaOH (5 N, 0.15 mL) was added and the mixture was stirred for 1hour. pH was adjusted to pH=5 with acetic acid and the mixture wasseparated between water and ethyl acetate. The organic phase was dried(Na₂SO₄) and solvent removed in vacuo to give the crude product whichwas purified on RP-HPLC, C-18 with acetonitrile/water gradient (75-95%)containing 0.1% TFA. To give pure4-[tert-Butoxycarbonylmethyl-(15-tert-butoxycarbonylpentadecanoyl)amino]benzoicacid) (53 mg, 19%)

HPLC-MS (fast grad): m/z=598 (M+Na), Rt=3.02 min.

Step 3:4-[tert-Butoxycarbonylmethyl(15-tert-butoxycarbonylpentadecanoyl)amino]benzoicacid 2,5-dioxopyrrolidin-1-yl ester

This compound was synthesized from4-[tert-Butoxycarbonylmethyl-(15-tert-butoxycarbonylpentadecanoyl)amino]benzoicacid) using a similar procedure as described in step 3 in generalprocedure A.

¹H-NMR (CDCl₃) δ 8.19 (d, 2H), 7.49 (d, 2H), 4.27 (s, 2H), 2.94 (s, 4H),2.17 (m, 4H), 1.57 (m, 4H), 1.46 (s, 9H), 1.44 (s, 9H), 1.30-1.15 (m,20H).

Step 4

4-[tert-Butoxycarbonylmethyl(15-tert-butoxycarbonylpentadecanoyl)amino]benzoicacid 2,5-dioxopyrrolidin-1-yl ester was reacted with A1,B1-diBoc desB30insulin as described in general procedure B.

HPLC-MS: m/z=1539 (m/4).

Example 9 N^(εB29)-(3-Carboxy-5-hexadecandioylaminobenzoyl) des(B30)insulin

5-Nitro-isophthalic acid mono t-Butyl ester

To a suspension of 5-nitro-isophthalic acid (5.0 g, 23.7 mmol) in drytoluene (100 ml) at 110° C. was added dimethylformamidedi-t-butylacetale (3.4 ml, 71 mmol) drop wise over 60 min. Heating wascontinued for 45 min and the reaction mixture was left at Rt over night.The precipitated starting material was removed by filtration. Thefiltrate was concentrated to give a yellow oil (7.88 g). This waspurified by flash chromatography using EtOAc/Hept 1:2 and EtOAc/Hept1:2+5% AcOH in two portions to give the title compound in 38% yield(2.38 g)

¹H-NMR (CDCl₃): δ 1.65 (s, 9H), 8.98 (s, 1H), 9.01 (s, 1H), 9.06 ppm (s,1H).

5-Amino-isophthalic acid mono t-Butyl ester

5-Nitro-isophthalic acid mono t-Butyl ester (2.38 g, 8.9 mmol) wasdissolved in EtOAc (50 ml). 10% Palladium on activated charcoal wasadded and the mixture was hydrogenated at 1 atm and room temperature.After 24 h, the mixture was filtered through a glass microfiber filterand the filtrate was concentrated to give the title compound as whitecrystals in 98% yield (2.07 g).

¹H-NMR (DMSO, d6): δ 1.54 (s, 9H), 5.59 (br, 2H, NH₂); 7.33 (s, 1H),7.37 (s, 1H), 7.60 ppm (s, 1H).

5-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acidmono-tert-butyl ester

To a solution of hexadecandioic acid mono t-butyl ester (100 mg, 0.29mmol) in dry DCM (2 ml), HOAt (44 mg, 0.29 mmol) and DCC (72 mg, 32mmol) was added. The mixture was stirred at 50° C. for 1 h. The oil bathwas removed. 5-Amino-isophthalic acid mono t-butyl ester (69 mg, 0.29)and DIPEA (0.07 ml, 0.32 mmol) was added. The mixture was stirred at RTover night under nitrogen. The yellow suspension was filtered and thefiltrate was concentrated. The residue was redissolved in EtOAc andextracted with 0.1 N HCl (2×), brine (1×), dried (Na₂SO₄) andconcentrated to give a white solid, which was purified twice by flashchromatography using EtOAc/Hept 1:2+5% AcOH. The title compound wasobtained in 86% yield (0.140 g) as an oil containing an impurity.

¹H NMR (400 MHz) δ: 1.25 (s, 22H) 1.42-1.45 (m, 9H) 1.53-1.65 (m, 11H)1.68-1.79 (m, 2H) 2.20 (s, 2H) 2.41 (s, 2H) 7.58 (brs, 1H) 8.35 (s, 1H)8.39 (s, 2H)

5-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid1-tert-butyl ester 3-(2,5-dioxo-pyrrolidin-1-yl)ester

5-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acidmono-tert-butyl ester (140 mg, 0.25 mmol) was dissolved in dry THF (3.5ml). TSTU (95 mg, 0.30 mmol) and DIPEA (70 ul, 0.30 mmol) were added.The mixture was stirred at RT under nitrogen over the week-end. Thereaction mixture was almost dry. EtOAc was added and the precipitate wasremoved by filtration. The filtrate was extracted with 0.1 N HCl (2×),brine (1×), dried (Na₂SO₄) and concentrated to give the title compoundas a syrup containing a trace of an impurity in a quantitative yield(0.165 mg).

1H NMR (400 MHz) δ: ppm 1.20-1.35 (m, 22H) 1.44 (s, 9H) 1.52-1.64 (m,11H) 1.68-1.79 (m, 2H) 2.12-2.24 (m, 2H) 2.34-2.42 (m, 2H) 2.91 (s, 4H)7.39 (s, 1H, NH) 8.34 (s, 1H) 8.43 (s, 1H) 8.54 (s, 1H)

N^(εB)29-(3-Carboxy-5-hexadecandioylamino-benzoyl) desB30 human insulin

A1N, B1N-diBoc desB30 human insulin (100 mg, 0.017 mmol) was acylatedwith 5-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid1-tert-butyl ester 3-(2,5-dioxo-pyrrolidin-1-yl) ester (15 mg, 0.022mmol) as described in general procedure B. The product was purified bypreparative HPLC and hydrolysed as described in general procedure B togive the title compound, 9 mg.

MALDI-MS (SA): 6130.8. anal. HPLC (neut, Alrg), 96.5% purity, rt 6.11min. (Column: C4 5μ 150×4.60 mm “phenomenex, Jupiter” Buffer A: 10 mMTris, 15 mM (NH₄)₂SO₄, pH 7.3, 20% CH₃CN in MQ water. Buffer B: 80%CH₃CN, 20% MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 10% B->20 min 50%B->21 min 50% B->23 min 10% B->30 min 10% B) anal. HPLC (acidic), 100%purity, rt 12.24 min (Column: C4 5μ 150×4.60 mm “phenomenex, Jupiter”Buffer A: 0.1% TFA, 10% CH₃CN, 89.9% MQ-water Buffer B: 0.1% TFA, 80%CH₃CN, 19.9% MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 20% B->17 min90% B->21 min 90% B->23 min 20% B->30 min 20% B.

Example 10 N^(εB29)-(3-Carboxy-5-octadecandioylaminobenzoyl) des(B30)human insulin

The title compound was prepared as described in example 9 usingoctadecanedioc acid monotertbutyl ester in stead of hexadecandioic acid.MALDI-MS (SA): 6160.9. anal. HPLC (neut), 96.0% purity, rt 8.93 min.(Column: C4 5μ 150×4.60 mm “phenomenex, Jupiter” Buffer A: 10 mM Tris,15 mM (NH₄)₂SO₄, pH 7.3, 20% CH₃CN in MQ water. Buffer B: 80% CH₃CN, 20%MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 10% B->20 min 50% B->21 min50% B->23 min 10% B->30 min 10% B). anal. HPLC (acidic), 100% purity, rt13.62 min (Column: C4 5μ 150×4.60 mm “phenomenex, Jupiter” Buffer A:0.1% TFA, 10% CH₃CN, 89.9% MQ-water Buffer B: 0.1% TFA, 80% CH₃CN, 19.9%MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 20% B->17 min 90% B->21 min90% B->23 min 20% B->30 min 20% B).

Example 11 Synthesis of N^(εB29)-ω-carboxypentadecanoyl-γ-L-glutamyl4-aminomethyl-benzoyl) desB30 human insulin

Mono-Tert-Butyl Hexadecandioate

Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250 ml)and the mixture was heated to reflux. N,N-dimethylformamidedi-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4hours. The mixture was refluxed overnight. The solvent was removed invacuo at 50° C., and the crude material was suspended in DCM/AcOEt (500ml, 1:1) and stirred for 15 min. The solids were collected by filtrationand triturated with DCM (200 ml). The filtrate was evaporated in vacuoto give crude mono-tert-butyl hexadecandioate, 30 grams. This materialwas suspended in DCM (50 ml), cooled with ice for 10 mins, and filtered.The solvent was removed in vacuo to leave 25 gram crude mono-tert-butylhexadecandioate, which was recrystallized from heptane (200 ml) to givemono-tert-butyl hexadecandioate, 15.9 g (33%). Alternatively torecrystallization, the mono-ester can be purified by silicachromatography in AcOEt/heptane.

¹H-NMR (CDCl₃) δ: 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44(s, 9H), 1.34-1.20 (m, 20H).

Succinimidyl Tert-Butyl Hexadecandioate

The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in THF (20 ml)and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2 ml, 7.0 mmol) andstirred overnight. The mixture was filtered, and the filtrate wasevaporated in vacuo. The residue was dissolved in AcOEt and washed twicewith cold 0.1 M HCl and water. Drying over MgSO₄ and evaporation invacuo gave succinimidyl tert-butyl hexadecandioate, 2.02 g (79%).

¹H-NMR (CDCl₃) δ: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t, 2H), 1.74 (p,2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H), 1.30-1.20 (m, 18H).

Tert-Butyl Hexadecandioyl-L-Glu-OtBu

Succinimidyl tert-butyl hexadecandioate (1 g, 2.27 mmol) was dissolvedDMF (15 ml) and treated with L-Glu-OtBu (0.51 g, 2.5 mmol) and DIEA(0.58 ml, 3.41 mmol) and the mixture was stirred overnight. The solventwas evaporated in vacuo, and the crude product was dissolved in AcOEt,and washed twice with 0.2M HCl, with water and brine. Drying over MgSO₄and evaporation in vacuo gave tert-butyl hexadecandioyl-L-Glu-OtBu, 1.2g (100%).

¹H-NMR (CDCl₃) δ: 6.25 (d, 1H), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m,4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.43-1.22(m, 18H).

Tert-Butyl Hexadecandioyl-L-Glu(OSu)-OtBu

Tert-butyl hexadecandioyl-L-Glu-OtBu (1.2 g, 2.27 mmol) was dissolved inTHF (15 ml) and treated with TSTU (0.82 g, 2.72 mmol) and DIEA (0.47 ml,2.72 mmol) and stirred overnight. The mixture was filtered, and thefiltrate was evaporated in vacuo. The residue was dissolved in AcOEt andwashed twice with cold 0.1 M HCl and water. Drying over MgSO₄ andevaporation in vacuo gave tert-butyl hexadecandioyl-L-Glu(OSu)-OtBu,1.30 g (92%).

¹H-NMR (CDCl₃) δ: 6.17 (d, 1H), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m,1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m,4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.33-1.21 (m, 20H).

Tert-butyl hexadecandioyl-L-Glu(NHCH₂PhCOOH)—OtBu

Tert-butyl hexadecandioyl-L-Glu(OSu)-OtBu (100 mg, 0.16 mmol) i DMF (1ml) was treated with 4-aminomethylbenzoic acid (27 mg, 0.18 mmol) andDIEA (41 μL, 0.24 mmol) and the mixture was stirred overnight. Thesolvent was evaporated and the residue was dissolved in AcOEt. Theorganic phase was washed with 2×0.2M HCl, water and brine.

Drying over MgSO₄ and evaporation in vacuo gave tert-butylhexadecandioyl-L-Glu(NHCH₂PhCOOH)—OtBu, 92 mg (87%).

¹H-NMR (CDCl₃) δ: 7.85 (d, 2H, J=8 Hz), 7.30 (d, 2H, J=8 Hz), 7.16 (t,1H), 7.43 (d, 1H), 4.50, (m, 3H), 2.39 (t, 2H), 2.29 (m, 1H), 2.25 (t,2H), 2.18 (t, 2H), 1.89 (m, 1H), 1.59 (m, 6H), 1.47 (s, 9H), 1.43 (s,9H), 1.25 (m, 20H).

Tert-butyl hexadecandioyl-L-Glu(NHCH₂PhCOOSu)-OtBu

tert-butyl hexadecandioyl-L-Glu(NHCH₂PhCOOH)—OtBu (92 mg, 0.14 mmol) wasdissolved in THF (1 ml) and treated with TSTU (50 mg, 0.17 mmol) andDIEA (29 μl, 0.177 mmol) and stirred overnight. The mixture wasfiltered, and the filtrate was evaporated in vacuo. The residue wasdissolved in AcOEt and washed twice with cold 0.1 M HCl and water.Drying over MgSO₄ and evaporation in vacuo gave tert-butylhexadecandioyl-L-Glu(NHCH₂PhCOOSu)-OtBu, 95 mg (90%).

¹H-NMR (CDCl₃) δ: 8.07 (d, 2H, J=8 Hz), 7.45 (d, 2H, J=8 Hz), 7.37 (t,1H), 6.38 (d, 1H), 4.53, (m, 2H), 4.40 (m, 1H), 2.89 (s, 4H), 2.32 (t,2H), 2.20 (m, 6H), 1.86 (m, 2H), 1.59 (m, 6H), 1.46 (s, 9H), 1.44 (s,9H), 1.25 (m, 20H).

N^(εB29)-ω-carboxypentadecanoyl-γ-L-glutamyl 4-aminomethyl-benzoyl)desB30 human insulin

Des(B30) human insulin (500 mg, 0.090 mmol) was dissolved in 100 mMNa₂CO₃ (6.5 ml, pH 10.2) at room temperature. Tert-butylhexadecandioyl-L-Glu(NHCH₂PhCOOSu)-OtBu (80 mg, 105 mmol) was dissolvedin acetonitrile (6.5 ml) and added to the insulin solution. After 30mins, 0.2 M methylamine (0.5 ml) was added. pH was adjusted by HCl to5.5 by use of 1 M HCl, and the isoelectric precipitate was collected bycentrifugation and dried in vacuo. The coupling yield was 84% (RP-HPLC,C4 column; Buffer A: 10% MeCN in 0.1% TFA-water, Buffer B: 80% MeCN in0.1% TFA-water; gradient 20% to 90% B in 16 minutes). The protectedproduct was dissolved in 95% TFA (15 ml), left 30 mins and evaporated invacuo. The crude product was dissolved in water and lyophilized.

N^(εB29)-hexadecandioyl-gamma-Glu-(4-aminomethyl-benzoyl) desB30 insulinwas purified by RP-HPLC on C4-column, buffer A: 20% EtOH+0.1% TFA,buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B, followed by HPLC onC4-column, buffer A: 10 mM Tris+15 mM ammonium sulphate in 20% EtOH, pH7.3, buffer B: 80% EtOH, gradient 15-60% B. The collected fractions weredesalted on Sep-Pak with 70% acetonitrile+0.1% TFA, neutralized byaddition of ammonia and freeze-dried. The unoptimized yield was 11 mg(2%). The purity as evaluated by HPLC was >98%. LCMS 6236,C₂₈₂H₄₁₈N₆₆O₈₂S₆ requires 6237.

Example 12 N^(εB29)-(3-Carboxy-4-(14-carboxytetradecyloxy)benzoyl)desB30 human insulin Step 1: 4-Hydroxy isophthalic acid dimethyl ester

4-Hydroxy isophthalic acid (5 g, 27.5 mmol) was dissolved in 100 mlmethanol, cooled to 0° C. under a flow of 4, and thionyl chloride wasadded over ca. 5 min. The reaction was stirred at 0° C. for 30 min andthen at room temperature for 1 h. The reaction was refluxed for 16 h.The solvent was removed under vacuum and the white solid was dissolvedin AcOEt (100 ml). The solution was washed with water (2×50 ml), driedover MgSO₄ and concentrated under vacuum to yield a white crystallinesolid (5.28 g, 92%).

¹H-NMR (CDCl₃, 300 MHz) δ: 11.20 (s, 1H), 8.57 (s, 1H), 8.12 (d, 1H),7.02 (d, 1H), 3.99 (s, 3H), 3.91 (s, 3H)

Step 2: 4-Hydroxyisophthalic acid 1-methyl ester

The compound was prepared in the manner described by Coutts, Ian G. C.;Edwards, Mark; Richards, David J. Synthesis 1981, 487. 4-Hydroxyisophthalic acid dimethyl ester (5.28 g, 25.1 mmol) was refluxed inpyridine under a flow of N₂. The reaction was monitored via TLC(20:10:1, heptane/AcOEt/AcOH) which indicated reaction completion after8 h. The majority of the pyridine was removed under vacuum, and AcOEt(100 ml) was added. The solution was washed with 0.5 M HCl (3×50 ml).The acidic washes were then extracted with AcOEt (100 ml). The twoorganic phases were pooled, dried (MgSO₄) and concentrated to yield awhite solid (4.85 g, 99%). The solid was recrystallized from toluene(200 ml) to yield white crystals (4.6 g, 92%).

¹H-NMR (DMSO, 400 MHz) δ: 8.39 (s, 1H), 8.05 (d, 1H), 7.07 (d, 1H), 3.84(s, 3H).

¹³C-NMR (DMSO, 400 MHz) δ 171.38, 165.59, 165.00, 136.26, 132.47,120.91, 118.11, 113.72, 52.40.

Step 3: 4-Hydroxy isophthalic acid 3-tert-butyl ester 1-methyl ester

4-Hydroxyisophthalic acid 1-methyl ester (2 g, 10.2 mmol) was heated to80° C. in toluene (50 ml) under a flow of N₂, and N,N-dimethylformamidedi-tert-butyl acetal (4.88 ml, 20.4 mmol) was added over 30 sec. Thereaction was stirred at 80° C., and monitored via TLC (20:10:1(Heptane/AcOEt/AcOH). After 1 h more N,N-dimethylformamide di-tert-butylacetal (4.88 ml, 20.4 mmol) was added, and once again after anadditional hour. The reaction was stirred at 80° C. for 1 h., and thesolvent was removed under vacuum. AcOEt (100 ml) was added and thesolution was washed with water (3×50 ml), dried over MgSO₄, andconcentrated under vacuum to yield light yellow crystals (2.64 g). Thesample was recrystallized from heptane (10 ml) to yield off-whitecrystals (1.26 g, 49%).

¹H-NMR (CDCl₃, 300 MHz) δ: 11.55 (s, 1H), 8.47 (s, 1H), 8.08 (d, 1H),6.98 (d, 1H), 3.91 (s, 3H), 1.64 (s, 9H), (also some contaminationsignals at 1.58 and 1.46).

Step 4: 15-Bromo-pentadecanoic acid tert-butyl ester

15-Bromo-pentadecanoic acid (5 g, 15.6 mmol) was heated to 70° C. undera flow of N₂ in toluene (50 ml). N,N-dimethylformamide di-tert-butylacetal (18.7 ml, 77.8 mmol) was added over 10 min. The reaction wasstirred at 55° C. for 16 h. The sample was concentrated under vacuum toa yellowish solid. The solid was dissolved in DCM (100 ml), washed withwater (2×40 ml) and dried over MgSO₄ to yield a white residue (5.35 g).The sample was recrystallized from ethanol (50 ml), by initially coolingslightly and filtering off the first precipitate. The filtrate was thencooled on an ice bath to form the desired crystals, which were filteredoff and dried to yield a white powder (1.37 g, 23%). Concentrating thefiltrate to 20 ml and cooling yielded another batch of crystals (0.99 g,15%).

HPLC-MS: m/z: 399+401 (M+23), Rt=7.04 min.

Step 5: 4-(14-tert-Butoxycarbonyl-tetradecyloxy)-isophthalic acid3-tert-butyl ester 1-methyl ester

15-Bromo-pentadecanoic acid tert-butyl ester (598 mg, 1.59 mmol),4-hydroxy isophthalic acid 3-tert-butyl ester 1-methyl ester (400 mg,1.59 mmol) and K₂CO₃ (329 mg, 2.38 mmol) were placed in a flask withacetonitrile (25 ml) and refluxed under N₂. The reaction was followedvia TLC (4:1 heptane/AcOEt). After 13 h the sample was concentratedunder vacuum to near dryness. AcOEt (50 ml) and water (25 ml) were addedto the residue. The phases were separated and the organic phase waswashed with water and brine (25 ml each), dried over MgSO₄ andconcentrated to yield an oil (841 mg, 97%).

HPLC-MS m/z: 571 (M+23) Rt=7.18 min.

Step 6: 4-(14-tert-Butoxycarbonyl tetradecyloxy)isophthalic acid3-tert-butyl ester

4-(14-tert-Butoxycarbonyl-tetradecyloxy)-isophthalic acid 3-tert-butylester 1-methyl ester (381 mg, 0.69 mmol) was dissolved in methanol (10ml). The solution was cooled to 0° C. and 4 N NaOH (1 ml) was added. Thesolution was allowed to warm to rt and more methanol (15 ml) was added.The reaction was stirred at rt for 30 min under N₂, and at reflux for 2h. The solution was cooled to 0° C. and 1 N HCl (1 ml) was added slowly.Water (25 ml) was added, and the solution was extracted with AcOEt (2×50ml). The organic phases were pooled and washed with 1:1 water/sat. NaCl,dried over MgSO₄, and concentrated under vacuum to yield an oily solid(326 mg, 89%)

HPLC-MS m/z: 557 (M+23), Rt=6.5 min.

Step 7: 4-(14-tert-Butoxycarbonyl-tetradecyloxy)isophthalic acid3-tert-butyl ester 1-(2,5-dioxo pyrrolidin-1-yl)ester

4-(14-tert-Butoxycarbonyl tetradecyloxy)isophthalic acid 3-tert-butylester (0.15 g, 0.28 mmol) was dissolved in THF (2 ml). DIEA (58 μl, 0.34mmol) was added, and the solution was cooled to 0° C. TSTU (0.10 g, 0.28mmol) was added. The reaction was stirred at 0° C. for 30 min and thenat rt for 16 h. The sample was concentrated under vacuum to neardryness. AcOEt (20 ml) was added and the solution was washed with 0.2 NHCl and sat. NaHCO₃ (3×5 ml each), dried over MgSO₄ and concentrated toyield a residue (0.18 g). The residue was purified by flashchromatography (silica: AcOEt/heptane 3:7 (0.5 l), 1:1 (0.2 l)) yieldingan oil with white solids (55 mg, 31%).

¹H-NMR (CDCl₃, 400 MHz) δ: 8.43 (s, 1H), 8.16 (d, 1H), 7.00 (d, 1H),4.10 (t, 2H), 2.90 (s, 4H), 2.20 (t, 2H), 1.86 (t, 2H), 1.58 (s,12H=9H+H₂O), 1.49 (m, 2H), 1.44 (s, 9H), 1.26 (m, 20H).

Step 8: N^(εB29)-(3-Carboxy-4-(14-carboxytetradecyloxy)benzoyl) desB30human insulin

4-(14-tert-Butoxycarbonyl-tetradecyloxy)isophthalic acid 3-tert-butylester 1-(2,5-dioxo pyrrolidin-1-yl)ester (13 mg, 0.021 mmol) was coupledto A1,B1-d-Boc-desB30 human insulin (123 mg, 0.021 mmol), and treatedwith TFA analogous to method used in general procedure B, and purifiedas such to yield 38 mg product.

MS: m/z: 1528.8, calculated: 6111.1: (M+4)/4).

Example 13 N^(εB29)-(3-Carboxy-5-(14-carboxytetradecyloxy)benzoyl)desB30 human insulin

Step 1: 5-(14-tert-Butoxycarbonyltetradecyloxy)isophthalic acid dimethylester

5-Hydroxy-isophthalic acid dimethyl ester (420 mg, 2 mmol),15-bromo-pentadecanoic acid tert-butyl ester (755 mg, 2 mmol) and K₂CO₃(415 mg, 3 mmol) were placed in a flask with acetonitrile (25 ml) andrefluxed under N₂. The reaction was followed by TLC (4:1 heptane/AcOEt),which indicated reaction completion after 6 h. The sample wasconcentrated to near dryness. AcOEt (50 ml) and water (25 ml) were addedto the residue. The phases were separated and the organic phase waswashed with water and brine (25 ml each), dried over MgSO₄ andconcentrated to yield a white crystalline solid (1.0 g, 100%).

HPLC-MS m/z: 529 (M+23), Rt=7.12 min.

¹H-NMR (CDCl₃, 300 MHz) δ: 8.26 (s, 1H), 7.74 (s, 2H), 4.03 (t, 2H),3.94 (s, 6H), 2.20 (t, 2H), 1.80 (m, 2H), 1.57 (m, 2H), 1.44 (s, 9H),1.26 (m, 20H).

Step 2: 5-(14-tert-Butoxycarbonyl-tetradecyloxy)isophthalic acidbis-(2,5-dioxo-pyrrolidin-1-yl)ester

5-(14-tert-Butoxycarbonyltetradecyloxy)isophthalic acid dimethyl ester(965 mg, 1.91 mmol) was dissolved in methanol (50 ml) at 50° C. under aflow of N₂. After 30 min 1 N NaOH (3.8 ml) was added and the solutionwas refluxed for 100 min. The solution was cooled and 1 N HCl (4.5 ml)was added. The sample was concentrated under vacuum, and the residue wastaken up in AcOEt (20 ml) and water (25 ml). The phases were separatedand the organic phase was washed with water (15 ml), died (MgSO₄) andconcentrated to a white solid (900 mg).

This residue was dissolved in THF (10 ml) and placed in an ice bath.DIEA (386 μl, 2.26 mmol) was added, followed by TSTU (675 mg, 1.88mmol). The reaction was stirred at 0° C. for 30 min and at rt for 16 h.The reaction mixture was concentrated under vacuum and AcOEt (50 ml) wasadded. The solution was washed with 0.2 N HCl (3×50 ml), water (50 ml)and sat. NaCl (30 ml), dried over MgSO₄, and concentrated under vacuumto yield an oil (1.12 g). The compound was purified by flashchromatography (silica: 2:3 AcOEt/heptane) to yield an oil containingsome crystals (330 mg).

Some of this compound (290 mg) was dissolved in methanol (15 ml) and 1 NNaOH (2.5 ml), and heated in an oil bath at 70° C. for 4.5 h. The samplewas evaporated to dryness, and AcOEt (25 ml), water (15 ml) and 1 N HCl(2.8 ml) were added. The phases were separated and the organic phase waswashed with water (15 ml), dried over MgSO₄, and concentrated to a whiteresidue (210 mg).

The residue was dissolved in THF (10 ml), DIPEA (75 μL, 0.44 mmol) wasadded, and the solution was cooled to 0° C. TSTU (173 mg, 0.48 mmol) wasadded and the reaction was stirred for 30 min at 0° C. and 16 h at rt.The reaction was evaporated to dryness, and AcOEt (25 ml) and 0.2 N HCl(26 ml) were added. The phases were separated and the organic phase waswashed with 0.2 N HCl (2×25 ml), sat. NaHCO₃ (3×25 ml) and sat. NaCl (25ml), dried over MgSO₄ and concentrated under vacuum. DCM was added andthe sample was concentrated to yield a residue. The residue washpurified by flash chromatography (silica: 7:3 AcOEt/heptane) to yield 90mg, which was again purified by flash chromatography (silica:AcOEt/heptane 4:6 (100 ml) then 7:3 (100 ml)) to yield a white residue(19 mg, 8%)

HPLC-MS (Gradient) m/z: 696 (M+23); Rt: 6.04 min.

¹H-NMR (CDCl₃, 400 MHz) δ: 8.46 (s, 1H), 7.90 (s, 2H), 4.06 (t, 2H),2.92 (s, 8H), 2.19 (t, 2H) 1.82 (m, 2H), 1.47 (m, 2H), 1.44 (s, 9H),1.26 (m, 20H).

Step 3: N^(εB29)-(3-Carboxy-5-(14-carboxy-tetradecyloxy)-benzoyl) desB30human insulin

The 5-(14-tert-Butoxycarbonyl-tetradecyloxy)isophthalic acidbis-(2,5-dioxo-pyrrolidin-1-yl)ester was reacted withA1-B1-di-Boc-des-B30-insulin, treated with TFA and purified analogous tothe general method B to yield the desired product.

Maldi-MS: m/z 6119.6: Calculated: 6111.1.

Example 14N^(εB29)-{4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB30human insulin Step 1: 4-Hydroxy-benzoic acid tert-butyl ester

4-Hydroxy-benzoic acid (2 g, 14.5 mmol) was heated to 80° C. in drytoluene (3 Å molecular sieves) under a flow of N₂. N,N-dimethylformamidedi-tert-butyl acetal (11.8 g, 57.92 mmol) is added over 5 min. Themixture was stirred at 80° C. for 50 min. The solution was washed withwater, sat. NaHCO₃ and sat. NaCl (15 ml each), dried over MgSO₄, andconcentrated to yield a yellow oil (2.92 g). Some of the oil (ca 1.7 g)was purified by küglerohr distillation (195°, 0.07 torr) to yield acolourless oil (1.18 g). ¹H-NMR indicated the product containedapproximately 30% of a by-product where the phenol group was protectedwith a tert-butyl group. The crude product was used in the subsequentreaction.

Step 2: 2-(10-Bromodecanoylamino)pentanedioic acid 5-benzyl ester1-tert-butyl ester

10-Bromodecanoic acid (3.20 g, 12.7 mmol) was dissolved in DMF (50 ml)and cooled to 0° C. under a flow of 4. DIEA (3.92 g, 30.3 mmol) and EDAC(2.56 g, 13.3 mmol) were added, and the solution was stirred at 0° for30 min. Glu-(OBn)-tBu HCl (2 g, 6.06 mmol) was added and the solutionwas stirred for 30 min at 0° C. and for 16 h at rt. The sample wasconcentrated under vacuum, and transferred to a separatory funnel withAcOEt (100 ml). The solution was washed once with water and twice with0.5 N NaOH and 5% AcOH (50 ml each), using some sat. NaCl and methanolto assist phase separation with the acidic washes. The organic phase wasdried over MgSO₄, and concentrated to yield an oil (2.84 g). The oil wasdissolved in 5 ml AcOEt and dispersed on a bed of silica in a glassfilter. Eluting with AcOEt (150 ml), and concentrating under vacuumyielded a light brown oil (1.65 g, 52%)

HPLC-MS m/z: 550 (M+23), Rt=5.19 min.

Step 3: 2-[10-(4-tert-Butoxycarbonylphenoxy)decanoylamino]pentanedioicacid 5-benzyl ester 1-tert-butyl ester

4-Hydroxy-benzoic acid tert-butyl ester (0.65 g, 3.08 mmol) wasdissolved in acetonitrile (7.5 ml) and added to2-(10-Bromodecanoylamino)pentanedioic acid 5-benzyl ester 1-tert-butylester (1.62 g, 3.08 mmol). Acetonitrile (90 ml) and K₂CO₃ (0.64 g, 4.62mmol) was added and the mixture was refluxed for 16 h under a flow ofN₂. The solvent was removed under vacuum. AcOEt (100 ml) was added andthe solution was washed with water (2×50 ml) using sat. NaCl andmethanol to aid phase separation, dried over MgSO₄, and concentrated toyield a light brown oil (2.18 g). The oil was purified by flashchromatography (silica, 95:5 DCM/AcOEt) to yield an oil (160 mg, 8%).

¹H-NMR (CDCl₃, 300 MHz) 8: (selected signals) 7.91 (d, 2H), 7.25 (s,5H), 6.87 (d, 2H), 6.06 (d, 1H), 5.11 (s, 2H), 4.52 (m, 1H), 3.98 (d,2H), 2.2-2.6 (m, 3H), 2.17 (t, 2H), 1.90-2.02 (m, 1H), 1.73-1.82 (m,2H), 1.58 (s, 9H).

HPLC-MS (Fastgrad) m/z: 640 (M+1), Rt=3.0 min.

Step 4: 2-[10-(4-tert-Butoxycarbonylphenoxy)decanoylamino]pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester

2-[10-(4-tert-Butoxycarbonylphenoxy)decanoylamino]pentanedioic acid5-benzyl ester 1-tert-butyl ester (160 mg, 0.25 mmol) was dissolved inTHF (10 ml) under a flow of N₂, and palladium (26 mg, 10% on carbon, 50%water) was added. The flask was evacuated and filled with N₂ four times,and a balloon filled with H₂ was connected to the system. The solutionwas stirred for 16 h at rt, and filtered through a bed of celite,washing with THF (100 ml). The filtrate was concentrated to yield thecarboxylic acid (190 mg). The crude product was dissolved in THF (5 ml)and cooled to 0° C. DIEA (64 μl, 0.375 mmol) and TSTU (0.09 g, 0.3 mmol)were added. The mixture was stirred at 0° C. for 1 h and at rt for 16 h.AcOEt (50 ml) was added, and the solution was washed with 0.2 M HCl(3×15 ml) and sat. NaHCO₃ (2×15 ml), dried over MgSO₄, and concentratedto yield an oil (167 mg). Purification by flash chromatography (2:1AcOEt/heptane) yielded a 107 mg of a colorless oil.

¹H-NMR (CDCl₃, 300 MHz) (selected signals) δ 7.92 (d, 2H), 6.87 (d, 2H),6.22 (d, 1H), 4.61 (m, 1H) 3.99 (t, 2H), 2.83 (s, 4H), 2.62-2.75 (m,2H), 2.30 (m, 1H), 2.22 (t, 2H), 2.10 (m, 1H), 1.58 (s, 9H).

Step 5:N^(εB29)-{4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB30human insulin

2-[10-(4-tert-Butoxycarbonylphenoxy)decanoylamino]pentanedioic acid1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester was coupled todesB30 human insulin in similar fashion as described in GeneralProcedure A. The intermediate product was purified by preparative HPLC(C₁₈-5 cm dia.) before treating with TFA. The product was purified bypreparative HPLC (C₄2 cm dia.) 15-65% acetonitrile) followed by ionexchange chromatography (Column: Amersham Resource Q-6 ml, Buffer A:0.24% w/w tris, 0.25% w/w ammonium acetate, 42.5% w/w ethanol, pH 7.5with acetic acid, Buffer B: 0.24% w/w tris, 2.5% w/w ammonium acetate,42.5% w/w ethanol, pH 7.5 with acetic acid.

HPLC-MS: m/z: 1532.6 (M+4)/4), calculated: 6126.

Example 15N^(εB29)-[3-Carboxy-5-(octadecandioyl-N-carboxyethylclycin)aminobenzoyl]desB30 human insulin

Step 1 Synthesis of 3-(Benzyloxycarbonylmethylamino)propionic acidtert-butyl ester

H-Gly-OBn, HCl (3.03 g, 15 mmol) was dissolved in dry DMF (15 ml) andcooled on an ice bath. TEA (2.10, 15 mmol) was added under precipitationof TEA-hydrochloride. The suspension was stirred for 5 min beforet-butyl acrylate (2.20 ml, 15 mmol) was added. The cooling bath wasallowed to reach RT slowly and stirring was continued under Nitrogen for2 days. The reaction mixture was filtered and the filtrate wasconcentrated. The residue, still containing DMF, was dissolved in EtOAcand washed with sat aq NaHCO₃ (2×) and water (1×). The organic layer wasfiltered before drying (Na₂SO₄) and concentration to give an oilPurification by chromatography or preparative HPLC gave3-(benzyloxycarbonylmethylamino)propionic acid tert-butyl as a clear oil(0.739 g, 17%).

¹H-NMR (CDCl₃) δ: ppm 1.46 (s, 9H) 2.50-2.61 (m, 2H) 2.82-2.99 (m, 2H)3.31 (s, 2H) 5.14 (s, 2H) 7.29-7.43 (m, 5H).

Step 2 Synthesis of17-[Benzyloxycarbonylmethyl-(2-tert-butoxycarbonyl-ethyl)-carbamoyl]-heptadecanoicacid tert-butyl ester

3-(Benzyloxycarbonylmethylamino)propionic acid tert-butyl ester (0.030g, 0.1 mmol) and octadecanedioic acid tert-butyl ester2,5-dioxo-pyrrolidin-1-yl ester (0.050 mg, 0.1 mmol) was suspended indry DMF (1 ml). HOAt (0.014 g, 0.1 mmol) and DIPEA (0.21 ml, 1.2 mmol)was added. The yellow reaction mixture was stirred under nitrogen for 42h. The reaction mixture was concentrated. The residue was redissolved inEtOAc and washed with 0.1 N HCl (2×), water (1×), dried (Na₂SO₄) andconcentrated to give17-[Benzyloxycarbonylmethyl-(2-tert-butoxycarbonyl-ethyl)-carbamoyl]-heptadecanoicacid tert-butyl ester in 85% yield (55 mg).

¹H-NMR (CDCl₃) δ: ppm 1.3 (m, 26H) 1.38 (s, 9H), 1.46 (s, 9H), 1.6 (m,4H), 2.2 (m, 2H), 2.35 (m, 2H), 2.65 (m, 2H), 2.85 (s, 2H) 3.65 (m, 2H),5.15 (s, 2H) 7.35 (m, 5H).

Step 3 Synthesis of17-[(2-tert-Butoxycarbonyl-ethyl)-carboxymethyl-carbamoyl]-heptadecanoicacid tert-butyl ester

17-[Benzyloxycarbonylmethyl-(2-tert-butoxycarbonyl-ethyl)-carbamoyl]-heptadecanoicacid tert-butyl ester (0.054 g, 0.08 mmol) was dissolved in THF (2 ml).10% Palladium on Charcoal was added and the mixture was hydrogenated at1 atm and RT over the week-end. The dry reaction mixture was dissolvedin EtOAc and filtered 3 times to remove the carbon. The filtrate wasconcentrated to give17-[(2-tert-Butoxycarbonyl-ethyl)-carboxymethyl-carbamoyl]-heptadecanoicacid tert-butyl ester in 80% yield (37 mg).

¹H-NMR (CDCl₃) δ: ppm 1.3 (m, 26H) 1.40 (s, 9H), 1.46 (s, 9H), 1.6 (m,4H), 1.75 (p, 2H), 2.2 (m, 2H), 2.35 (m, 2H), 2.63 (m, 2H), 2.83 (s,2H).

Step 4 Synthesis of5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid mono-tert-butyl ester

17-[(2-tert-Butoxycarbonyl-ethyl)-carboxymethyl-carbamoyl]-heptadecanoicacid tert-butyl ester (0.130 g) was dissolved in dry DCM (5 ml). HOAt(0.36 mg) and DIC (0.045 ml) was added the mixture was refluxed for 1 hunder nitrogen. The reaction mixture was cooled to room temperature and5-amino-isophthalic acid mono t-Butyl ester (60 mg) was added. Afterstirring for 1 h DIPEA (0.050 ml) was added, the orange reaction mixtureturns yellow. After stirring for 2 days, the reaction mixture wasconcentrated. The residue was redissolved in EtOAc and extracted with0.1 N HCl (2×) and brine (1×), dried (Na₂SO₄), and concentrated to givean sirup, which solidifies on standing.5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid mono-tert-butyl ester was obtained in a quantitative yield (214mg), contaminated with an impurity. HPLC/MS 775 (M), rt 7.64 min.

Step 5 Synthesis of5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid 1-tert-butyl ester 3-(2,5-dioxo-pyrrolidin-1-yl)ester

5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid mono-tert-butyl ester (214 mg) was dissolved in dry THF and TSTU(0.105 mg) and DIPEA (0.1 ml) was added. The mixture was stirred at roomtemperature under nitrogen. After 20 h the reaction mixture wasconcentrated. The residue was redissolved in EtOAc and filteret. Thefiltrate was extracted with 0.1 N HCl (2×) and brine (1×), dried(Na₂SO₄) and concentrated to give5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid 1-tert-butyl ester 3-(2,5-dioxo-pyrrolidin-1-yl)ester as a yellowsirup in 90% yield (218 mg).

Step 6 Synthesis ofN^(εB29)-[3-Carboxy-5-(octadecandioyl-N-carboxyethylglycin)amino-benzoyl]desB30insulin

5-{2-[(2-tert-Butoxycarbonyl-ethyl)-(17-tert-butoxycarbonyl-heptadecanoyl)-amino]-acetylamino}-isophthalicacid 1-tert-butyl ester 3-(2,5-dioxo-pyrrolidin-1-yl)ester was reactedwith A1,B1-diBoc insulin as described in general procedure B. Theproduct was purified by preparative HPLC to give the title compound.Over all yield for coupling and hydrolysis, 18% (21 mg).

MALDI-MS (SA): 6288.8. anal. HPLC (neut), 93.8.5% purity, rt 10.22 min.(Column: C4 5μ 150×4.60 mm “phenomerex, Jupiter” Buffer A: 10 mM Tris,15 mM (NH₄)₂SO₄, pH 7.3, 20% CH₃CN in MQ water. Buffer B: 80% CH₃CN, 20%MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 5% B->20 min 55% B->22 min80% B->24 min 80% B->25 min 5% B) anal. HPLC (acidic), 100% purity, rt11.694 min (Column: C4 5μ 150×4.60 mm “phenomerex, Jupiter” Buffer A:0.1% TFA, 10% CH₃CN, 89.9% MQ-water Buffer B: 0.1% TFA, 80% CH₃CN, 19.9%MQ-water, Flow: 1.5 ml/min; Gradient: 0 min 20% B->17 min 80% B->22 min80% B->23 min 20% B->30 min 20% B.

Example 16 General Procedure A, Acylation Using desB30 Human Insulin)N^(εB29{)3-[(3,5-Bis-carboxymethoxy-benzyl)-(15-carboxypentadecanoyl)amino]-propionyldesB30 human insulin Step 1: Synthesis of Hexadecanedioic acidmono-(4-methoxy-benzyl)ester

Hexadecandioic acid (2 g, 7 mmol) was dissolved in dry NMP (25 mL).Diisopropylamin (1.2 mL, 7 mmol) and 4-methoxybenzyl chloride (0.95 mL,7 mmol) was added, followed by NaI (0.52 g, 3.5 mmol). The mixture washeated to 80° C. for 1 hour, poured into water (100 mL) and filtered.The precipate was washed with dichloromethane (150 mL), thedichoromethane phase was dried (Na₂SO₄) and solvent removed in vacuo toyield the crude product, which was recrystallized several times fromheptane to yield hexadecanedioic acid mono-(4-methoxy-benzyl)ester.

¹H NMR (CDCl₃): δ 7.28 (d, 2H), 6.87 (d, 2H), 5.02 (s, 2H), 3.78 (s,3H), 2.31 (m, 4H), 1.60 (m, 4H), 1.20 (m, 20H).

Step 2: Synthesis of(3-Tert-Butoxycarbonylmethoxy-5-formylphenoxy)acetic acid tert-butylester

4,5 dihydroxybenzaldehyde (2.5 g, 18.1 mmol) was dissolved in NMP (120mL). Potassium carbonate (10 g, 72.4 mmol) was added, followed bytert-butyl bromoacetate.

The mixture was stirred at room temperature, under nitrogen overnight.The reaction was filtered and separated between diethyl ether (400 mL)and water (400 mL). The organic phase was dried (Na₂SO₄) and solventremoved in vacuo to yield the crude product as an oil which solidifiesby standing. The crude product was used in the next step without furtherpurification.

HPLC-MS: m/z=(389, M+Na); R_(t)=4.50 min.

¹H NMR (CDCl₃): δ 9.85 (s, 1H), 7.00 (s, 2H), 6.75 (s, 1H), 4.58 (s,4H), 1.44 (2, 18H)

Step 3: Synthesis of3-(3,5-Bis-tert-butoxycarbonylmethoxybenzylamino)propionic acid

Beta-alanine (0.5 g, 5.68 mmol) was dissolved in methanol (20 mL).(3-Tert-Butoxycarbonylmethoxy-5-formylphenoxy)acetic acid tert-butylester (2.08 g, 5.58 mmol) was dissolved in methanol (2 mL) and added.The mixture was heated to reflux for 1 hour and allowed to cool to roomtemperature. Sodium cyanoborohydride (282 mg, 4.54 mmol) was added andthe mixture stirred at room temperature, after 1 hour acetic acid wasadded (2 mL) and the mixture was stirred for an additional hour beforebeing poured into water (50 mL) and stirred overnight. The water phasewas washed with ethyl acetate (2×50 mL). The organic phase was dried(Na₂SO₄) and solvent removed in vacuo to yield the crude product. Thecrude product was used in the next step without further purification.

HPLC-MS: m/z=(440, M+Na); R_(t)=3.24 min.

Step 4: Synthesis of15-[(3,5-Bis-tert-butoxycarbonylmethoxybenzyl)-(2-carboxyethyl)carbamoyl]pentadecanoicacid 4-methoxybenzyl ester

Hexadecanedioic acid mono-(4-methoxy-benzyl)ester (0.4 g, 0.98 mmol) wasdissolved in ethyl acetate (10 mL).N-Ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.187 g,0.98 mmol) and 1-hydroxy-7-azabenzotriazole (0.134 g, 0.98 mmol) wasadded and the mixture was stirred at 50° C. for 1 hour. After cooling toroom temperature, diisopropylethylamin (0.50 mL, 2.95 mmol) was addedfollowed by 3-(3,5-Bis-tert-butoxycarbonylmethoxybenzylamino)propionicacid (0.432 g, 0.98 mmol). The mixture was stirred overnight undernitrogen at room temperature. The mixture was separated between ethylacetate (200 mL) and water (2×100 mL). The organic phase was dried(Na₂SO₄), solvent removed in vacuo. The crude product was purified byRP-HPLC on C18-column, buffer A: 0.1% TFA, buffer B: MeCN+0.1% TFA;gradient 80-100% B to yield the title compound.

HPLC-MS: m/z=(547, M+Na); R_(t)=6.17 min.

Step 5: Synthesis of15-{(3,5-Bis-tert-butoxycarbonylmethoxybenzyl)-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethyl]carbamoyl}pentadecanoicacid 4-methoxy-benzyl ester

15-[(3,5-Bis-tert-butoxycarbonylmethoxybenzyl)-(2-carboxyethyl)carbamoyl]pentadecanoicacid 4-methoxybenzyl ester (190 mg, 0.23 mmol) was dissolved in THF (5mL). The mixture was cooled with an ice bath. Diisopropylethylamin(0.047 mL, 0.28 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (83mg, 0.28 mmol) was added. The mixture was stirred under nitrogen at 0°C. After 30 minutes the ice cooling was removed and the mixture wasstirred for an additional 3 hours. Solvent removed in vacuo. The crudeproduct was dissolved in ethyl acetate (50 mL), washed with aqueousphosphate buffer (pH=5.5) (3×25 mL). The organic phase was dried(Na₂SO₄), solvent removed in vacuo to yield the title compound (163 mg)which was used in subsequent step.

HPLC-MS: m/z=924; R_(t)=6.5 min.

Step 6: Synthesis ofN^(εB29){3-[(3,5-Bis-carboxymethoxy-benzyl)-(15-carboxy-pentadecanoyl)-amino]-propionyldesB30 human insulin

DesB30 human insulin (742 mg, 0.13 mmol) was dissolved in aqueous Na₂CO₃(100 mM, 14.7 mL).15-{(3,5-Bis-tert-butoxycarbonylmethoxybenzyl)-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethyl]carbamoyl}pentadecanoicacid 4-methoxy-benzyl ester (120 mg, 0.13 mmol) was dissolved inacetonitrile (7.4 mL) and added. The mixture was stirred very slowly for1 hour at RT. pH was adjusted to 5.5 with aqueous HCl (1N) and thesuspension was allowed to stand for 10 minutes at 0° C. The precipitatewas isolated by centrifugation and treated with mixture of p-cresol(0.750 mL) and TFA (14.25 mL) for 10 minutes. Poured into ice cooleddiethylether (30 mL), and the crude product was isolated by centrifugeand purified with RP-HPLC on a Waters Prep LC2000, on C18, 5 cm×20 cm,flow 20 ml/min using acetonitrile/water 15-55% gradient containing 0.1%TFA. Fractions containing product was collected and lyophilized. To thelyophilized material was added water (7.2 mL) and pH adjusted to 8.98with 1 N+0.1 N NaOH. The pH was adjusted back to 5.2-5.5 with 0.1 N HCl.The product precipitated, isolated by centrifugation and lyophilized togive the title compound.

HPLC-MS: m/z=1257 (m/5), R_(t)=3.27 min.

Example 17N^(εB29)-3-[4′-(2-Carboxyethyl)biphenyl-4-yl]propionyl-γ-L-glutamyldesB30 insulin

Step 1: Synthesis of tert-butyl 3-(4-bromophenyl)propionate

3-(4-Bromophenyl)propionic acid (1.0 g, 4.4 mmol) was dissolved intoluene (15 ml) and treated with N,N-dimethylformamide ditert-butylacetal (1.8 g, 8.7 mmol). The mixture was heated to 90° C. for 5 hours,and then treated with more N,N-dimethylformamide ditert-butyl acetal(1.8 g, 8.7 mmol). The mixture was left at 90° C. overnight. Ethylacetate was added (25 ml) and the organic phase was washed with 2×0.1 MHCl, 2×5% Na₂CO₃ and water. Drying over MgSO₄ and evaporation in vacuogave tert-butyl 3-(4-bromophenyl)propionate, 0.735 g (59%).

¹H-NMR (CDCl₃) δ: 7.47 (d, 2H), 7.08 (d, 2H), 2.87 (t, 2H), 2.52 (t,2H), 1.42 (s, 9H).

Step 2: Synthesis of tert-butyl3-[4′-(2-Carboxy-ethyl)-biphenyl-4-yl]-propionate

Tert-butyl 3-(4-bromophenyl)propionate (433 mg, 1.52 mmol) inacetonitrile-water (3:1, 13 ml) under argon atmosphere was treated with4-(2-carboxyethyl)benzeneboronic acid (294 mg, 1.52 mmol), K₂CO₃ (251mg, 1.82 mmol) and (Ph₃P)₄Pd (87 mg, 73 μmol), and the stirred mixturewas heated at 90° C. for 4 hours. Excess ethyl acetate and 2 M HCl wasadded and the organic phase was washed with 2×2 M HCl and 2× water.Drying over MgSO₄ and evaporation in vacuo gave the crude product, whichwas purified by chromatography on silica column eluted with ethylacetate/hexane/acetic acid 50:50:1 to provide tert-butyl3-[4′-(2-Carboxy-ethyl)-biphenyl-4-yl]-propionate, 330 mg (62%).

¹H-NMR (CDCl₃) δ: 7.50 (dd, 4H), 7.26 (dd, 4H), 3.00 (t, 2H), 2.94 (t,2H), 2.72 (t, 2H), 2.57 (t, 2H), 1.42 (s, 9H).

Step 3: Synthesis of tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate

Tert-butyl 3-[4′-(2-Carboxy-ethyl)-biphenyl-4-yl]-propionate (330 mg,0.93 mmol) was dissolved in THF (5 ml) and treated with TSTU (336 mg,1.12 mmol) and DIEA (191 μL, 1.12 mmol), and the mixture was stirred atroom temperature overnight. The mixture was filtered, the solvent wasevaporated in vacuo, and the crude product was dissolved in ethylacetate, and washed with 2×0.1 M HCl, 2×5% Na₂CO₃ and water. Drying overMgSO₄ and evaporation in vacuo gave tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate, 374 mg(89%).

¹H-NMR (CDCl₃) δ: 7.50 (dd, 4H), 7.27 (dd, 4H), 3.10 (t, 2H), 2.95 (m,4H), 2.84 (s, 4H), 2.57 (t, 2H), 1.42 (s, 9H).

Step 4: Synthesis of tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-glutamylα-tert-butyl ester

Tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate (100mg, 0.22 mmol) was dissolved in DMF (1.0 ml) and treated with L-Glu-OtBu(50 mg, 0.25 mmol) and DIEA (56 μL, 0.33 mmol) and stirred at roomtemperature overnight. The solvent was removed in vacuo, and the residuewas dissolved in ethyl acetate and washed with 2×0.2 M HCl, water andbrine. Drying over MgSO₄ and evaporation in vacuo gave tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-glutamylα-tert-butyl ester, 119 mg (100%).

¹H-NMR (CDCl₃) δ: 7.48 (dd, 4H), 7.24 (dd, 4H), 6.30 (bd, 1H), 4.54 (m,1H), 2.95 (m, 4H), 2.56 (m, 4H), 2.31 (m, 2H), 2.16 (m, 1H), 1.86 (m,1H), 1.44 (s, 9H), 1.42 (s, 9H).

Step 5: Synthesis of tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-glutamyl-γ-O-succinimidylα-tert-butyl ester

Tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-Glu-OtBu(120 mg, 0.22 mmol) in THF (2 ml) and reacted with TSTU (80 mg, 0.27mmol) and DIEA (46 μL, 0.27 mmol) as described in step 3 above toprovide tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-glutamyl-γ-O-succinimidylα-tert-butyl ester, 136 mg (96%).

¹H-NMR (CDCl₃) δ: 7.50 (dd, 4H), 7.25 (dd, 4H), 6.21 (d, 1H), 4.60 (m,1H), 3.00 (t, 2H), 2.94 (t, 2H), 2.78 (s, 4H), 2.56 (m, 6H), 2.36 (m,1H), 2.04 (m, 1H), 1.46 (s, 9H), 1.43 (s, 9H).

Step 5: Synthesis ofN^(εB29)-3-[4′-(2-Carboxy-ethyl)-biphenyl-4-yl]-propionyl-γ-L-glutamyldesB30 insulin

DesB30 human insulin (500 mg, 88 μmol) was reacted with tert-butyl3-[4′-(2-O-succinimidyl-carboxy-ethyl)-biphenyl-4-yl]-propionate-L-glutamyl-γ-O-succinimidylα-tert-butyl ester (67 mg, 105 μmol), and the product was isolated,deprotected and HPLC-purified as described for example 11. LCMS: 6114.0,C₂₇₆H₃₉₉N₆₅O₈₁S₆ requires 6116.0.

Example 18 General Procedure A, Acylation Using desB30 Human InsulinN^(εB29)-ω-carboxypentadecanoy-(4-aminomethylbenzoyl)-γ-L-glutamyldesB30 human insulin -Glu-desB30 insulin Step 1: Synthesis of4-[(15-tert-Butoxycarbonylpentadecanoylamino)methyl]benzoic acid

To 4-(Aminomethyl)benzoic acid (0.2 g, 1.32 mmol) was added in NMP (5mL). Hexadecanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-ylester (0.58 g, 1.32 mmol) was added and the mixture was stirred at roomtemperature overnight. The mixture was poured into water (100 mL), theprecipitate was isolated by filtration and dried in vacuo. The crudematerial was recrystallized from toluene to give4-[(15-tert-butoxycarbonylpentadecanoylamino)methyl]benzoic acid (413mg).

¹H NMR (DMSO-d₆): δ 12.77 (br s, 1H), 8.36 (t, 1H), 7.87 (d, 2H), 7.32(d, 2H), 4.32 (d, 2H), 2.15 (q, 4H), 1.48 (m, 4H), 1.38 (s, 9H),1.28-1.18 (br s, 20H)

Step 2: Synthesis of(S)-2-{4-[(15-tert-Butoxycarbonyl-pentadecanoylamino)-methyl]-benzoylamino}-pentanedioicacid 1-tert-butyl ester

4-[(15-tert-butoxycarbonylpentadecanoylamino)methyl]benzoic acid (413mg, 0.868 mmol) was dissolved in THF (5 mL), the solution was cooledwith an icebath. DIPEA (0.33 mL, 1.91 mmol) and TSTU (314 mg, 1.04 mmol)were added. The mixture was stirred under nitrogen while cooling wasmaintained. After 30 minutes the icebath was removed and the mixture wasstirred for additional 3 hours at room temperature. The mixture wasdiluted with NMP (5 mL) and H-GluOtBu (0.21 g, 1.04 mmol) was added, themixture was stirred overnight at room temperature. The mixture wasseparated between ethyl acetate (100 mL) and water (100 mL), the organicphase dried (Na₂SO₄) and solvent removed in vacuo. The crude materialwas purified on silica using DCM/ethanol (90:10) to give(S)-2-{4-[(15-tert-Butoxycarbonyl-pentadecanoylamino)-methyl]-benzoylamino}-pentanedioicacid 1-tert-butyl ester.

¹H NMR (CDCl₃): δ 7.63 (d, 2H), 7.20 (m, 3H), 6.52 (t, 1H), 4.62 (m,1H), 4.40 (d, 2H), 2.50 (m, 2H), 2.30-2.10 (m, 6H), 1.70-1.55 (m, 4H),1.50 (s, 9H), 1.45 (s, 9H), 1.35-1.20 (m, 20H)

Step 3: Synthesis ofN^(εB29)-ω-carboxypentadecanoyl-(4-aminomethylbenzoyl)-γ-L-glutamyldesB30 human insulin

The compound was prepared similar as described in step 3 and step 4 ingeneral procedure B using(S)-2-{4-[(15-tert-Butoxycarbonyl-pentadecanoylamino)-methyl]-benzoylamino}-pentanedioicacid 1-tert-butyl ester.

Example 19N^(εB29)(4-{[(2-Carboxy-ethyl)-(15-carboxy-pentadecanoyl)amino]methyl}benzoyl)-γ-D-glutamyldesB30 human insulin Step 1: Synthesis of resin bound Fmoc-D-Glu-OtBu

1 g of polystyrene resin functionalized with a 2-chlorotrityl chloridelinker (1.4 mmol/g) was vortexed with NMP (10 mL) and1,2-dichloropropane (10 mL) for 1 hour. The resin was filtered andwashed with dichloromethane (20 mL). Fmoc-D-Glu-OtBu (596 mg, 1.4 mmol)was dissolved together with diisopropylethylamine (0.96 mL, 5.6 mmol) indichloromethane (20 mL) and added to the resin. After shaking thesuspension for 2 hours at 25° C., the resin was isolated by filtrationand washed with NMP (2×20 mL)

Step 2: Synthesis of resin bound 4-Formylbenzoyl-D-Glu-OtBu

To the above resin bound Fmoc-D-Glu-OtBu was treated with a 20% solutionof piperidine in NMP (2×20 mL in 2×5 min), after the resin was drainedand washed with NMP (6×20 mL). NMP (10 mL) and diisopropylethylamin(0.96 mL) was added to the resin. 4-formylbenzoic acid (0.841 g, 5.6mmol) and 1-hydroxybenzotriazole (0.757 g, 5.6 mmol) were dissolved inNMP (10 mL), followed by diisopropylcarbodiimide (0.867 mL, 5.6 mmol)and stirred for The for 10 minutes before added to the resin. Themixture was shaken for 2 hours at 25° C. followed by filtration andwashing of the resin with N-methyl-2-pyrrolidinone (3×20 mL).

Step 3: Synthesis of resin bound4-[(2-tertbutoxycarbonylethylamino)-methyl]benzoyl-D-Glu-OtBu

The above resin bound 4-Formylbenzoyl-D-Glu-OtBu was treated withtert-Butyl beta-alanine hydrochloride (0.902 g, 5 mmol) anddiisopropylamine (0.856 mL, 5 mmol) in a mixture of NMP andtrimethylorthoformate (1:1 10 mL) and glacial acetic acid (1 mL) for 1hour at 25° C. Sodium cyanoborohydride (314 mg, 5 mmol) was dissolved ina mixture of N-methyl-2-pyrrolidinone and methanol (1:1, 5 mL) andadded. The mixture was vortexed at 25° C. for 4 hours followed byfiltration and washing with a mixture of NMP and methanol (1:1, 2×20mL), NMP (3×20 mL) and a mixture of 1,2-dichloropropane anddiisopropylethylamine (7:1, 2×20 mL).

Step 4: Synthesis of resin bound4-{[(2-tert-butoxycarbonylethyl)-(15-tertbutoxycarbonylpentadecanoyl)-amino]-methyl}benzoyl-D-Glu-OtBu

The above resin bound4-[(2-tertbutoxycarbonylethylamino)-methyl]benzoyl-D-Glu-OtBu was addedsolution of hexadecanedioic acid mono-tert-butyl ester (685 mg, 2 mmol)in NMP, 1,2-dichloropropane, DIPEA (4.5:4.5:1, 10 mL) followed by asolution of bromo-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBrop) (0.93 g, 2 mmol.) dissolved in 1,2-dichloropropane (10 mL). Themixture was vortexed at 50° C. for 3 hours followed by filtration andwashing with NMP (4×20 mL) and DCM (10×20 mL).

Step 5: Synthesis of 4-{[(2-tert-butoxycarbonylethyl)-(15-tertbutoxycarbonylpentadecanoyl)-amino]-methyl}benzoyl-D-Glu-OtBu

The resin bound 4-{[(2-tert-butoxycarbonylethyl)-(15-tertbutoxycarbonylpentadecanoyl)-amino]-methyl}benzoyl-D-Glu-OtBu wastreated with DCM containing 1% TFA (2×20 mL, 2×10 min). After filtrationthe DCM/TFA fraction were collected and washed with NaHCO₃ 5% (20 mL).The organic phase was dried (Na2SO4), solvent removed in vacuo and crudematerial was purified on silicagel column eluted with DCM/EtOH 95:5 togive 4-{[(2-tert-butoxycarbonylethyl)-(15-tertbutoxycarbonylpentadecanoyl)-amino]-methyl}benzoyl-D-Glu-OtBu

¹H-NMR (CDCl₃): δ7.80 (dd, 2H), 7.30-7.08 (m, 3H), 4.72-4.60 (m, 3H),3.60-3.50 (m, 2H), 2.57-2.40 (m, 5H), 2.37-2.04 (m, 5H), 1.70-1.53 (m,4H), 1.50 (s, 9H), 1.45 (m, 18H), 1.25 (m, 20H). HPLC-MS (Method 50-99):m/z=811 (M+Na); R_(t)=2.32 min.

Step 6: Synthesis of Synthesis of(R)-2-(4-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-decanoyl)-amino]-methyl}-benzoylamino)-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxo-pyrrolidin-1-yl)ester

The compound was prepared similar as described in step 3 in generalprocedure A using(R)-2-{4-[(15-tert-Butoxycarbonyl-pentadecanoylamino)-methyl]-benzoylamino}-pentanedioicacid 1-tert-butyl ester.

HPLC-MS (Method 50-99): m/z=(908, M+Na); R_(t)=2.37 min.

Step 7: Synthesis ofN^(εB29)(4-{[(2-Carboxy-ethyl)-(15-carboxy-pentadecanoyl)amino]methyl}benzoyl)-γ-D-glutamyldesB30 human insulin

Des-B30 human insulin (386 mg, 0.068 mmol) was dissolved in DMSO (3.5mL) together with triethylamin (0.094 mL, 0.677 mmol).(R)-2-(4-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-decanoyl)-amino]-methyl}-benzoylamino)-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxo-pyrrolidin-1-yl)ester (60 mg, 0.068mmol) was dissolved in THF (1 mL) and added. The mixture was stirred atroom temperature for 30 minutes. Cooled on an icebath and diluted withMilli-Q water (7 mL). The pH was adjusted to 5.5 with 1 N HCl, whichlead to precipitation. The tube was centrifuged and the solvent wasdecanted from the solid. The solid was washed once with Milli-Q water (7mL), and centrifuged again. Solvent was decanted from the solid and tothe solid was added TFA (10 mL). The mixture was stirred for 30 minutesand poured into diethylether (35 mL) and centrifuged, after drying invacuo The crude material was purified Äkta purifier similar to what hasbeen described above.

HPLC-MS (Method Sciex): m/z=1578 (m/4), 1262 (m/5); R_(t)=3.38 min

Example 20N^(εB29)-4-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}benzoyl)-γ-L-glutamyldesB30 human insulin General Procedure A, Acylation Using desB30 HumanInsulin Step 1: Synthesis of(S)-2-(4-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-decanoyl)-amino]-methyl}-benzoylamino)-pentanedioicacid 1-tert-butyl ester

To H-Glu-OtBu, HCl (30.5 mg, 0.128 mmol) in DMF (1 mL) was added DIPEA(0.022 mL, 0.128 mmol),4-{[(2-tert-Butoxycarbonylethyl)-(15-tert-butoxycarbonylpentadecanoyl)amino]methyl}benzoic acid 2,5-dioxopyrrolidin-1-yl ester(45 mg, 0.064 mmol) was dissolved in DMF (1 mL) and added. The mixturewas stirred under nitrogen at room temperature overnight, separatedbetween ethylacetate and water. The organic phase was dried (MgSO₄) andsolvent removed in vacuo.

HPLC-MS (Method fast grad): m/z=(789, M+1); R_(t)=2.39 min.

Step 2: Synthesis of(S)-2-(4-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-decanoyl)-amino]-methyl}-benzoylamino)-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxo-pyrrolidin-1-yl)ester

The compound was prepared similar as described in step 3 in generalprocedure A using(S)-2-{4-[(15-tert-Butoxycarbonyl-pentadecanoylamino)-methyl]-benzoylamino}-pentanedioicacid 1-tert-butyl ester.

HPLC-MS (Method fast grad): m/z=(908, M+Na); R_(t)=2.51 min.

Step 3: Synthesis ofN^(εB29)-4-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}benzoyl)-γ-L-glutamyldesB30 human insulin

The compound was prepared similar as described in step 4 in generalprocedure B using(S)-2-(4-{[(2-tert-Butoxycarbonyl-ethyl)-(15-tert-butoxycarbonyl-decanoyl)-amino]-methyl}-benzoylamino)-pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxo-pyrrolidin-1-yl)ester.

Example 21 General Procedure AN^(εB29)-{4-[2-(4-carboxymethylphenyl)ethyl]phenyl}acetyl-γ-L-glutamyldesB30 human insulin Step 1: Synthesis of2-(2-{4-[2-(4-tert-Butoxycarbonylmethyl-phenyl)-ethyl]-phenyl}-acetylamino)-pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester

The compound was prepared similar as described in example 11 step 1 to 4(for tert-butyl hexadecandioyl-L-Glu (OSu)-OtBu) starting from4,4-dimethylbis(Phenylacetic acid) (purchased from Sigma-Aldrich Libraryof Rare Chemicals).

Step 2: Synthesis ofN^(εB29)-{4-[2-(4-carboxymethylphenyl)ethyl]phenyl}acetyl-γ-L-glutamyldesB30 human insulin

2-(2-{4-[2-(4-tert-Butoxycarbonylmethyl-phenyl)-ethyl]-phenyl}-acetylamino)-pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester was reactedwith Human des(B30) insulin followed by TFA treatment similar asdescribed in general procedure A. Purification by RP-HPLC was performedon a Gilson 215 system using a SP 250/21 Nucleosil 300-7 C4 column and awater/acetonitrile 30-80% gradient containing 0.1% TFA. Fractionscontaining product were collected and lyophilized.

MALDI-MS: (SA); m/z: 6117.57. Acidic HPLC: Rt=9.61 min; 98.8% purity.Run time 30 min. Column: C4 5μ 150×4_(—)60 mm “phenomenex, Jupiter”.A-Buffer: 0.1% TFA, 99.9% MQ-water, B-buffer: 0.1% TFA, 99.9%Acetonitrile. Flow: 1.5 ml/min. Gradient: 0-17 min, 20-90% B, 17-21 min90% B, 21-23 min 90-20% B, 23-30 min 20% B. Neutral HPLC: Rt=4.20 min;99.44% purity. Run time: 30 min. Column: C4 5μ 150×4_(—)60 mm“phenomenex, Jupiter”. A-buffer: 10 mM Tris, 15 mM (NH₄)₂SO₄, 20%acetonitrile in Mili Q water, pH 7.3 B-buffer: 20.0% MQ-water inacetonitrile. Flow: 1.5 ml/min, 1-20 min: 10-50% B, 20-22 min: 50-60% B,22-23 min: 60-10% B, 23-30 min 10% B30-31 min 10% B flow: 0.15 ml/min.214 nm.

Example 22 N^(εB29)-(3-carboxy-4-hexdecandioylaminobenzoyl) desB30 humaninsulin Step 1: 4-Nitro-isophthalic acid

Potassium permanganate (13.07 g) was dissolved in water (80 ml) in aflask fitted with a thermometer and a reflux condenser. 4-Nitro-m-xylene(2.23 ml) was added. The mixture was cautiously heated to 85° C. Coolingto maintain the reaction mixture at 85° C. was not necessary. After 20min. the mixture was refluxed gently for 3 h (the purple colour haddisappeared and the mixture was almost black). The warm mixture wasfiltered through celite. The cold filtrate was acidified withconcentrated sulfuric acid and a milky suspension was obtained.Extraction with EtOAc (3×). The combined organic layers was dried(Na₂SO₄) and concentrated to give a white crystalline compound.Purification by flash chromatography using EtOAc/Heptane/AcOH 10:10:1 aseluent gave a white crystalline compound in 44% yield (1.55 g).

¹H-NMR (DMSO-d₆) δ: 8.07 (d, 1H) 8.26 (d, 1H) 8.33 ppm (s, 1H).

Step 2: 3-tert-Butyl 4-Nitro-isophthalate

4-Nitro-isophthalic acid (1.0 g) was dissolved in hot toluene (30 ml)and DMF (2 ml). Dimethylformamid-di-t-butyl acetate (3.4 ml) was addeddropwise over 1 h min at 100° C. Stirring at 100° C. was continued for135 min. The cold reaction mixture was concentrated to give a crudemixture of starting material, 4-nitro-isophthalic acid di-tert-butylester, 1-tert-butyl 4-nitro-isophthalate and 3-tert-butyl4-nitro-isophthalate. Purification by flash chromatography usingEtOAc/Heptane/AcOH 5:15:1 or DCM/AcOH 20:1 resulted in isolation of3-tert-Butyl 4-Nitro-isophthalate contaminated with 1-tert-Butyl4-Nitro-isophthalate (10:1). The isomers were determined byNOE-experiments.

¹H-NMR (CDCl₃) δ: 1.55 (s 9H), 7.89 (d, 1H) 8.32 (d, 1H) 8.49 ppm (s,1H). HPLC-MS: 268 (M+1).

Step 3: 3-tert-Butyl 4-amino-isophthalate

3-tert-Butyl 4-Nitro-isophthalate (100 mg) was dissolved in EtOAc (3 ml)and 10% Pd/C was added. The mixture was hydrogenated at 1 atm for 2 h.The mixture was filtered and concentrated to give the title compound asa white foam in quantitative yield (90 mg).

¹H-NMR (CDCl₃) δ: 1.60 (s 9H), 6.32 (d, 1H) 7.86 (d, 1H) 8.58 ppm (s,1H). HPLC-MS: 238 (M+1).

Step 4: 4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid3-tert-butyl ester

Hexadecanedioic acid mono-t-Bu ester (60 mg) was dissolved in dry THF (1ml). N,N,N′,N′-Tetramethylfluorformamidiniumhexafluorophosphate (46 mg)was added. The mixture was stirred at RT under Nitrogen. A fineprecipitate was observed after a while. After 75 min 3-tert-Butyl4-amino-isophthalate (9:1 mixture, 45 mg) and DIPEA (0.05 ml) was added.After 5 days the mixture was concentrated. The residue was dissolved inEtOAc and extracted with 0.1 M HCl (2×), washed with brine (1×), dried(Na₂SO₄) and concentrated to give a sirup, which was purified by flashchromatography using EtOAc/Hept/AcOH 4:16:1 to give the productcontaminated with hexadecanedioic acid mono-t-Bu ester 1:4. (63 mg).

HPLC-MS: 562 (M+1).

Step 5: 4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid3-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester

The compound was prepared similar as described in example 1 (generalprocedure A) step 3 using4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid3-tert-butyl ester instead.

HPLC-MS: 659 (M+1).

Step 6: N^(εB29)-(3-carboxy-4-hexdecandioylaminobenzoyl) desB30 humaninsulin

4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-isophthalic acid3-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester was reacted withA1,B1,BOC,BOC-human desB30 insulin followed by TFA treatment similar asdescribed in general procedure B. Purification by RP-HPLC was performedon a Gilson 215 system using a SP 250/21 Nucleosil 300-7 C4 column and awater/acetonitril 20-80% gradient containing 0.1% TFA. Fractionscontaining product were collected and lyophilized.

MALDI-MS: (SA); m/z: 6140.3. Acidic HPLC: Rt=11.27 min; 83.4% purity.Rub time: 30 min. Column: C4 5μ 150×4_(—)60 mm “phenomenex, Jupiter”.A-Buffer: 0.1% TFA, 99.9% MQ-water, B-buffer: 0.1% TFA, 99.9%Acetonitrile. Flow: 1.5 ml/min. Gradient: 0-17 min, 20-90% B, 17-21 min90% B, 21-23 min 90-20% B, 23-30 min 20% B. Neutral HPLC: Rt=9.10 min;92.6% purity: Run time: 30 min Column: C4 5μ 150×4_(—)60 mm “phenomenex,Jupiter”. A-buffer: 10 mM Tris, 15 mM (NH₄)₂SO₄, 20% acetonitrile inMili Q water, pH 7.3 B-buffer: 20.0% MQ-water in acetonitrile. Flow: 1.5ml/min 1-20 min: 5% B til 50% B, 20-22 min: 50-60% B, 22-23 min: 60% Btil 5% B, 23-30 min 5 μl 0% B 30-31 min 0-5% B, flow: 0.15 ml/min. 214nm.

Example 23 N^(εB29)-10-(4-carboxyphenylsulfanyl)decanoyl-γ-L-glutamyldesB30 human insulin

Step 1: 4-(9-Methoxycarbonyl nonylsulfanyl)benzoic acid4-Mercaptobenzoic acid (2.0 g, 13 mmol) was placed in THF (25 ml). DIEA(3.7 g, 28.5 mmol) was added followed by a solution of methyl10-bromodecanoate (3.44 g, 13 mmol) in THF (10 ml). After 1 h thesolvent was removed under vacuum to yield a slurry, which was stored atrt for 3 days. AcOEt (100 ml) and 1 N HCl (50 ml) were added, but theprecipitate did not dissolve very well. Sat. NaCl was added and thenmethanol in order to aid phase separation. The aqueous phase wasremoved, and DCM was added to the organic phase, but the precipitatesstill did not dissolve. The organic phase was concentrated under vacuumand dried with toluene by adding and evaporating twice. Drying undervacuum yielded a white solid (4.4 g, quantitative yield).

HPLC-MS (fast grad) m/z: 361 (M+23), R_(t)=2.34 min.

¹H-NMR (DMSO, 300 MHz) δ 12.86 (br, 1H), 7.84 (d, 2H), 7.37 (d, 2H),3.57 (s, 3H), 3.03 (t, 2H), 2.28 (t, 2H), 1.33-1.69 (m, 6H), 1.24 (s,8H).

Step 2: 4-(9-Methoxycarbonyl nonylsulfanyl)benzoic acid tert-butyl ester4-(9-Methoxycarbonyl nonylsulfanyl)benzoic acid (4.4 g, 13 mmol) wassuspended in dry toluene (150 ml), under N₂. The mixture was refluxedand a solution of N,N-dimethylformamide di-tert-butyl acetal (7.93 g, 39mmol) in toluene (50 ml) was added over ca. 15 min. After refluxing 16h, the reaction was allowed to cool and some precipitation occurred. TLC(1:2 AcOEt/heptane) indicated ca. 50% completion. The reaction washeated to 70° C. and another portion of N,N-dimethylformamidedi-tert-butyl acetal (7.93 g, 39 mmol) in toluene (50 ml) was added over1.5 h. After stirring an additional hour at 70° C., the sample wasconcentrated under vacuum to yield an brown oil. Purification by flashchromatography (15 cm×40 mm dia., 1:2 AcOEt/heptane) yielded a yellowoil (3.65 g, 71%)

HPLC-MS (fast grad) m/z: 417 (M+23), R_(t)=3.03 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.88 (d, 2H), 7.26 (d, 2H), 3.66 (s, 3H), 2.96(t, 2H), 2.30 (t, 2H), 1.60-1.75 (m, 4H), 1.59 (s, 9H), 1.43 (t-br, 2H),1.29 (s, 8H).

Step 3: 4-(9-Carboxynonylsulfanyl)benzoic acid tert-butyl ester4-(9-Methoxycarbonyl nonylsulfanyl)benzoic acid tert-butyl ester (2.46g, 6.2 mmol) was dissolved in THF (25 ml). 1 N NaOH (6.2 ml, 6.2 mmol)was added and the mixture was stirred under N₂ for 1 d. 1 N HCl (6.5 ml)diluted with water (100 ml) was added, and then AcOEt (100 ml) wasadded. The organic phase was dried over MgSO₄ and concentrated undervacuum to yield a white solid (2.5 g).

HPLC-MS (fast grad) m/z: 403 (M+23), R_(t)=2.69 min.

¹H-NMR (DMSO, 300 MHz) δ 11.99 (br, 1H), 7.79 (d, 2H), 7.36 (d, 2H),3.03 (t, 2H), 2.18 (t, 2H), 1.60 (m, 2H), 1.53 (s, 9H), 1.32-1.51 (m,4H), 1.24 (s, 8H).

Step 4:(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 5-benzyl ester 1-tert-butyl ester-4-(9-Carboxynonylsulfanyl)benzoicacid tert-butyl ester (1 g, 2.6 mmol) was dissolved in THF (10 ml), EDAC(0.53 g, 2.8 mmol), HOBt (0.39 g, 2.9 mmol) and DIEA (1.0 g, 7.8 mmol)were added. The solution was stirred under N₂. A precipitate formed, andDMF (10 ml) was added and a clear solution was obtained. After stirringat rt for 30 min, H-Glu(OBzl)-OtBu (0.87 g, 2.6 mmol) was added. Thesolution was stirred under N₂ for 16 h at rt. The sample wasconcentrated under vacuum. AcOEt (100 ml) was added, and the solutionwas washed with water (50 ml), and 0.2 M HCl (2×50 ml), and dried overMgSO₄, and concentrated under vacuum to yield a light oil. Purificationby flash chromatography (15 cm×40 mm dia., 1:2 AcOEt/heptane) yielded acolorless oil (401+525 mg, 54% yield).

HPLC-MS (50-99) m/z: 656 (M+1), R_(t)=2.44 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.87 (d, 2H), 7.35 (s, 5H), 7.25 (d, 2H), 6.07(d, 1H), 4.52 (m, 1H), 2.95 (t, 2H), 2.31-2.35 (m, 2H), 2.11-2.27 (m,3H), 1.88-2.03 (m, 1H), 1.53-1.71 (m, 13H), 1.46 (s, 9H), 1.42 (m, 2H),1.28 (m, 8H).

Step 5:(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 1-tert-butylester(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 5-benzyl ester 1-tert-butyl ester (385 mg, 0.587 mmol) wasdissolved in THF. 1 N NaOH (587 μl, 0.587 mmol) was added and thesolution was stirred for 16 h at rt under N₂. The solvent hadevaporated, so more THF (3 ml) was added. AcOEt (40 ml) and dilute HCl(1 ml 1N HCl in 25 ml water) was added. The phases were separated andthe aqueous phase was extracted with AcOEt (15 ml). The organic phaseswere pooled and washed with sat. NaCl, dried over MgSO₄. The solutionwas concentrated under vacuum to yield a light brown oil. The oil waspurified by flash chromatography (7.5 cm×40 mm dia., 20:20:1AcOEt/heptane/AcOH) and after concentrating the appropriate fractionsunder vacuum, toluene was added and removed under vacuum a few times toremove residual AcOH to yield a colorless oil (160 mg, 48% yield).

HPLC-MS (50-99) m/z: 566 (M+1), R_(t)=1.65 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.87 (d, 2H), 7.26 (d, 2H), 6.25 (d, 1H), 4.52(m, 1H), 2.96 (t, 2H), 2.38-2.47 (m, 2H), 2.12-2.30 (m, 3H), 1.82-1.99(m, 1H), 1.60-1.75 (m, 4H), 1.58 (s, 9H), 1.47 (s, 9H), 1.36-1.45 (m,2H), 1.28 (s, 8H).

Step 6:(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 5-tert-butyl ester1-(2,5-dioxopyrrolidin-1-y1l)ester(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 1-tert-butyl ester (156 mg, 0.276 mmol) was dissolved in THF (3ml). DIEA (47 μl, 0.276 mmol) was added and the solution was cooled to0° C. TSTU (99 mg, 0.276 mmol) was added and the solution was stirredunder nitrogen at 0° C. for 30 min, and then at rt for 16 h. The samplewas concentrated under vacuum and partitioned between AcOEt and 0.2 NHCl. The organic phase was dried over MgSO₄ and concentrated undervacuum to yield a residue (194 mg).

HPLC-MS (50-99) m/z: 686 (M+23), R_(t)=1.46 min.

¹H-NMR (DMSO, 400 MHz) δ 8.12 (d, 1H), 7.79 (d, 2H), 7.36 (d, 2H), 4.16(m, 1H), 3.03 (t, 2H), 2.81 (s, 4H), 2.61-2.78 (m, 4H), 2.10 (t, 2H),1.99-2.07 (m, 1H), 1.80-1.94 (m, 1H), 1.42-1.66 (m, 11H), 1.38 (s, 9H),1.24 (s, 8H). (singlet at 2.69, ca. 2H possible impurity).

Step 7: N^(εB29)-10-(4-carboxyphenylsulfanyl)decanoyl-γ-L-glutamyldesB30 human insulin

General Coupling and Deprotection Method A:

Des-B30 insulin (125 mg, 0.022 mmol) was dissolved by adding 100 mMNa₂CO₃ (1.5 ml) and acetonitrile (1.5 ml) in a 10 ml round bottom-flask.(S)-2-[10-(4-tert-Butoxycarbonylphenylsulfanyl)decanoylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-y1l)ester (14.5 mg,0.022 mmol) was added in acetonitrile (750 ul) and Na₂CO₃ (750 ul) wasadded so the final solution was 50:50 100 mM Na₂CO₃/acetonitrile. Thesolution was stirred at rt for 1 h. The solution was transferred to a 15ml centrifuge tube, washing with Milli-Q water (6 ml). The solution wascooled on ice, and the pH was adjusted to 5.1 by adding 1 N HCl, whichlead to precipitation. The tube was centrifuged at 5000 rpm for 10 minat 10° C. The solvent was decanted from the solid. 95:5 TFA/water (2.5ml) was added to the solid. The solution was poured into a flask,washing with more 95:5 TFA/water (2.5 ml). The solution was stirred for30 min at rt, and concentrated under vacuum. DCM was added and removedtwice, and the flask was dried under vacuum at rt. The product waspurified by preparative HPLC (2 cm dia. C₁₈ column,acetonitrile/water/0.05% TFA). The relevant fractions were pooled (twobatches) and diluted 1:1 with water. The solutions were cooled on ice,and precipitation was induced by adjusting the pH to ca. 5 with 1 NNaOH. The samples were centrifuged (5000 rpm, 10 min, 5° C.). The liquidwas decanted off and the pellets were lyophilized to yield a white solid(30 mg+5 mg).

HPLC-MS (Sciex) m/z: 1536.7 (M/4+1=1536.5), R_(t)=3.2 min.

HPLC (neutral) R_(t)=5.60 min.

Example 24 N^(εB29)-11-(4-carboxyphenylsulfanyl)undecanoyl-γ-L-glutamyldesB30 human insulin

The following steps in the synthesis ofN^(εB29)-11-(4-carboxyphenylsulfanyl)undecanoyl-γ-L-glutamyl desB30human insulin were performed in a similar fashion as those described forN^(εB29)-10-(4-carboxyphenylsulfanyl)decanoyl-γ-L-glutamyl desB30 humaninsulin.

Step 1: 4-(10-Methoxycarbonyldecylsulfanyl)benzoic acid

HPLC-MS (fast grad) m/z: 375 (M+23), R_(t)=2.44 min.

¹H-NMR (DMSO, 300 MHz) δ 12.85 (br, 1H), 7.83 (d, 2H), 7.36 (d, 2H),3.57 (s, 3H), 3.03 (t, 2H), 2.28 (t, 2H), 1.60 (m, 2H), 1.58 (m, 2H),1.40 (m, 2H), 1.23 (s, 10H).

Step 2: 4-(10-Methoxycarbonyldecylsulfanyl)benzoic acid tert-butyl ester

¹H-NMR (CDCl₃, 300 MHz) δ 7.87 (d, 2H), 7.26 (d, 2H), 3.67 (s, 3H), 2.96(t, 2H), 2.30 (t, 2H), 1.57-1.75 (m, 13H), 1.45 (m, 2H), 1.28 (s, 10H).

Step 3: 4-(10-Carboxydecylsulfanyl)benzoic acid tert-butyl ester

HPLC-MS (50-99) m/z: 417 (M+23), R_(t)=1.82 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.87 (d, 2H), 7.26 (d, 2H), 2.96 (t, 2H), 2.35(t, 2H), 1.55-1.74 (m, 13H), 1.43 (m, 2H), 1.28 (s, 10H).

Step 4:(S)-2-[11-(4-tert-Butoxycarbonylphenylsulfanyl)undecanoylamino]pentanedioicacid 5-benzyl ester 1-tert-butyl ester

¹H-NMR (CDCl₃, 300 MHz) δ 7.86 (d, 2H), 7.35 (s, 5H), 7.26 (d, 2H), 6.06(d, 1H), 5.11 (s, 2H), 4.52 (m, 1H), 2.96 (t, 2H), 2.39 (m, 2H),2.11-2.28 (m, 3H), 1.88-2.06 (m, 1H), 1.60-1.73 (m, 4H), 1.58 (s, 9H),1.46 (s, 9H), 1.35-1.43 (m, 2H), 1.26 (s, 10H).

Step 5:(S)-2-[11-(4-tert-Butoxycarbonylphenylsulfanyl)undecanoylamino]pentanedioicacid 1-tert-butyl ester

HPLC-MS (50-99) m/z: 602 (M+23), R_(t)=1.80 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.87 (d, 2H), 7.26 (d, 2H), 6.25 (d, 1H), 4.52(m, 1H), 2.96 (t, 2H), 2.40 (m, 2H), 2.14-2.31 (m, 3H), 1.80-1.98 (m,1H), 1.60-1.75 (m, 4H), 1.58 (s, 9H), 1.47 (s, 9H), 1.36-1.45 (m, 2H),1.26 (s, 10H).

Step 6:(S)-2-[11-(4-tert-Butoxycarbonylphenylsulfanyl)undecanoylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester

HPLC-MS (50-99) m/z: 699 (M+23), R_(t)=2.05 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.87 (d, 2H), 7.26 (d, 2H), 6.19 (d, 1H), 4.60(m, 1H), 2.96 (t, 2H), 2.84 (s, 4H), 2.68-2.78 (m, 1H), 2.56-2.67 (m,1H), 2.27-2.39 (m, 1H), 2.22 (t, 2H), 2.01-2.14 (m, 1H), 1.59-1.75 (m,4H), 1.58 (s, 9H), 1.48 (s, 9H), 1.37-1.46 (m, 2H), 1.28 (s, 10H).

Step 7: B29N(eps)-11-(4-carboxy-phenylsulfanyl)undecanoyl gamma-GludesB30 insulin

HPLC-MS (Sciex) m/z: 1539.8 (M/4+1=1540) Rt: 3.5 min.

HPLC (neutral) R_(t)=5.93.

Example 25 N^(εB29)-10-(4-Carboxyphenoxy)decanoyl beta-Asp desB30insulin

Step 1: 4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester4-Hydroxybenzoic acid tert-butyl ester (500 mg, 2.57 mmol) and10-bromodecanoic acid methyl ester (683 mg, 2.57 mmol) were dissolved inacetonitrile, and K₂CO₃ was added. The mixture was refluxed undernitrogen for 16 h. The solids were filtered off, and the filtrate wasconcentrated under vacuum. The residue was dissolved in AcOEt (50 ml)and water (25 ml). The phases were separated and the organic phase wasdried over MgSO₄ and concentrated to yield a colorless oil (874 mg, 90%yield).

HPLC-MS (50-99) m/z: 402 (M+23), R_(t)=1.65 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 3.67(s, 3H), 2.31 (t, 2H), 1.78 (m, 2H), 1.62 (m, 2H), 1.58 (s, 9H), 1.45(m, 2H), 1.31 (s, 8H).

Step 2: 4-(9-Carboxynonyloxy)benzoic acid tert-butylester-4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester (858mg, 2.27 mmol) was dissolved in THF (5 ml). 1 N NaOH (2.27 ml) was addedand the mixture was covered lightly with a rubber septum, and stirredfor 16 h at rt. AcOEt (40 ml) and 1.05 eq 1N HCl in water (25 ml) wereadded. The phases were separated, and the organic phase was dried overMgSO₄, and concentrated under vacuum to yield a white solid (781 mg, 95%yield).

HPLC-MS (50-99) m/z: 387 (M+23), R_(t)=1.46 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 2.35(t, 2H), 1.75 (m, 2H), 1.64 (m, 2H), 1.58 (s, 9H), 1.45 (m, 2H), 1.32(s, 8H).

Step 3: 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester 4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester (779mg, 2.14 mmol) was dissolved in THF (15 ml), and DIEA (366 μl, 2.14mmol) was added. The solution was cooled to 0° C., and placed undernitrogen. TSTU (768 mg, 2.14 mmol) was added. The solution was stirredat 0° C. for 30 min then at rt for 16 h. The sample was concentratedunder vacuum. AcOEt (40 ml) was added, and the solution was washed with0.2 N HCl (2×25 ml), dried over MgSO₄, and concentrated under vacuum toyield a yellowish solid. The solid was recrystallized from AcOEt toyield a white powder (276 mg, 28%).

HPLC-MS (50-99) m/z: 484 (M+23), R_(t)=1.71 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 2.83(s, 4H), 2.61 (t, 2H), 1.67-1.88 (m, 4H), 1.58 (s, 9H), 1.27-1.52 (m,10H).

Step 4: (S)-2-[10-(4-tert-butoxycarbonylphenoxy)decanoylamino]succinicacid 1-tert-butyl ester4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester (264 mg, 0.57 mmol) was dissolved in DMF (2.5 ml).H-Asp-OtBu was added, and more DMF (2.5 ml). After 1 h DIEA (1 eq., 98ul) was added, and after 30 min more, DMF (5 ml) was added. There wasstill a lot of undissolved solids. After 1d at rt the solvent wasremoved under vacuum. AcOEt (40 ml) was added and the solution waswashed with 0.2 N HCl (2×25 ml), dried over MgSO₄ and concentrated undervacuum to yield an opaque oil (283 mg, 92% yield).

HPLC-MS (50-99) m/z: 558 (M+23), R_(t)=1.57 min.

¹H-NMR (DMSO, 300 MHz) δ 12.40 (br, 1H), 8.14 (d, 1H), 7.82, (d, 2H),6.99 (d, 2H), 4.44 (q, 1H), 4.02 (t, 2H), 2.52-2.92 (m, 2H), 2.08 (t,2H), 1.71 (t, 2H), 1.20-1.55 (m, 28H).

Step 5: (S)-2-[10-(4-tert-Butoxycarbonylphenoxy)decanoylamino]succinicacid 4-tert-butyl ester1-(2,5-dioxopyrrolidin-1-yl)ester(S)-2-[10-(4-tert-butoxycarbonylphenoxy)decanoylamino]succinicacid 1-tert-butyl ester (261 mg, 0.49 mmol) was dissolved in THF (5 ml).The solution was cooled to 0° C., and DIEA (100 μl, 0.59 mmol) and TSTU(175 mg, 0.49 mmol) were added. The mixture was stirred for 16 h in asmall ice bath, such that it could warm to RT after ca. 1 h. The samplewas concentrated under vacuum. AcOEt (40 ml) was added, and the solutionwas washed with 0.2 N HCl (2×25 ml), dried over MgSO₄, and concentratedunder vacuum to yield a colorless oil containing some white solid. Theproduct was purified by flash chromatography (35 g silica, 400 ml 1:1AcOEt/heptane and 100 ml 7:3 AcOEt/heptane) to yield a white solid (200mg, 65% yield).

HPLC-MS (Sciex) m/z: 633 (M+1), R_(t)=6.09 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.91 (d, 2H), 6.87 (d, 2H), 6.51 (d, 1H), 4.83(m, 1H), 3.99 (t, 2H), 3.24 (m, 2H), 2.83 (s, 4H), 2.25 (t, 2H), 1.78(m, 2H), 1.62-1.70 (m, 2H), 1.58 (s, 9H), 1.47 (s, 9H), 1.36-1.46 (m,2H), 1.31 (s, 8H).

Step 6: N^(εB29)-10-(4-Carboxyphenoxy)decanoyl beta-Asp desB30 insulin

-   -   The compound was prepared using the General Coupling and        Deprotection Method A to yield a white solid (26 mg and 8 mg).

HPLC-MS (Sciex) m/z: 1529.3 (M/4+1=1529), Rt=3.4 min.

HPLC (neutral) R_(t)=5.31 min.

Example 26 N^(εB29)-11-(4-Carboxy-phenoxy)undecanoyl γ-L-glutamyl desB30insulin

The following steps in the synthesis ofN^(εB29)-11-(4-Carboxy-phenoxy)undecanoyl γ-L-glutamyl desB30 insulinwere performed in a similar fashion as those described forN^(εB29)-10-(4-Carboxyphenoxy)decanoyl beta-Asp desB30 insulin.

Step 1: 4-(10-Methoxycarbonyldecyloxy)benzoic acid tert-butyl ester

HPLC-MS (50-99) m/z: 415 (M+23), R_(t)=2.31 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 3.67(s, 3H), 2.30 (t, 2H), 1.79 (m, 2H), 1.62 (m, 2H), 1.58 (s, 9H), 1.43(m, 2H), 1.30 (s, 10H).

Step 2: 4-(10-Carboxydecyloxy)benzoic acid tert-butyl ester

HPLC-MS (fast grad) m/z: 401 (M+23), R_(t)=2.71 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 3.98 (t, 2H), 2.34(t, 2H), 1.78 (m, 2H), 1.62 (m, 2H), 1.58 (s, 9H), 1.44 (m, 2H), 1.30(s, 10H).

Step 3: 4-[10-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)decyloxy]benzoic acidtert-butyl ester

HPLC-MS (50-99) m/z: 498 (M+23), R_(t)=1.89 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.92 (d, 2H), 6.88 (d, 2H), 3.99 (t, 2H), 2.84(s, 4H), 2.60 (t, 2H), 1.66-1.90 (m, 4H), 1.58 (s, 9H), 1.43 (m, 2H),1.32 (s, 10H).

Step 4:(S)-2-[11-(4-tert-Butoxycarbonylphenoxy)undecanoylamino]pentanedioicacid 1-tert-butyl ester

HPLC-MS (50-99) m/z: 564 (M+1), R_(t)=1.68 min.

¹H-NMR (CDCl₃, 400 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 4.50 (br, 1H),3.98 (t, 2H), 2.38 (br, 2H), 2.24 (t, 2H), 2.04-2.20 (br, 1H), 1.82-1.98(br, 1H), 1.69-1.82 (m, 2H), 1.59-1.67 (m, 2H), 1.57 (s, 9H), 1.38-1.50(m, 11H), 1.29 (s, 10H).

Step 5:(S)-2-[11-(4-tert-Butoxycarbonylphenoxy)undecanoylamino]pentanedioicacid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl)ester

HPLC-MS (50-99) m/z: 683 (M+23), R_(t)=1.91 min.

¹H-NMR (CDCl₃, 300 MHz) δ 7.92 (d, 2H), 6.87 (d, 2H), 6.20 (d, 1H), 4.60(m, 1H), 3.99 (t, 2H), 2.84 (s, 4H), 2.54-2.80 (m, 2H), 2.26-2.42 (m,1H), 2.22 (t, 2H), 2.04-2.15 (m, 1H), 1.72-1.88 (m, 2H), 1.60-1.70 (m,2H), 1.58 (s, 9H), 1.48 (s, 9H), 1.39-1.46 (m, 2H), 1.30 (s, 10H).

Step 6: N^(εB29)-11-(4-Carboxy-phenoxy)undecanoyl γ-L-glutamyl desB30insulin

HPLC-MS (Sciex) m/z: 1536.4 (M/4+1=1536.0), R_(t)=3.92 min.

HPLC (neutral) R_(t)=5.18 min.

Example 27 Insulin Receptor Binding of the Insulin Derivatives of theInvention

The affinity of the insulin derivatives of the invention for the humaninsulin receptor was determined by a SPA assay (Scintillation ProximityAssay) microtiterplate antibody capture assay. SPA-PVT antibody-bindingbeads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) weremixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodiumchloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a singlePackard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a1:5000 diluted purified recombinant human insulin receptor—exon 11, anamount of a stock solution of A14 Tyr[¹²⁵I]-human insulin correspondingto 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. Atotal of 100 μl was then added and a dilution series is made fromappropriate samples. To the dilution series was then added 100 μl ofreagent mix and the samples were incubated for 16 hours while gentlyshaken. The phases were the then separated by centrifugation for 1 minand the plates counted in a Topcounter. The binding data were fittedusing the nonlinear regression algorithm in the GraphPad Prism 2.01(GraphPad Software, San Diego, Calif.). Receptor binding (% of humanProduct insulin) Human insulin 100N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB30 18human insulinN^(εB29)-[N—(HOOC(CH₂)₁₃CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB30 28human insulin N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-para-C₆H₄CO]desB30 17 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-ortho-C₆H₄CO] desB30 11human insulin N^(εB29)-[N—(HOOC(CH₂)₁₄CO)-γ-Glutamyl-N—CH₂-para-C₆H₄CO]desB30 14 human insulinN^(εB29)-(3-Carboxy-5-hexadecandioylamino-benzoyl)desB30 insulin 12N^(εB29)-[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB30 15human insulinN^(εB29)-[(5-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}furan-2-13 carbonyl)desB30 human insulinN^(εB29)-(3-Carboxy-5-octadecandioylamino-benzoyl) des(B30) humaninsulin 19N^(εB29)-{4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB3046 human insulin N^(εB29)-10-(4-carboxy-phenylsulfanyl)decanoyl-γ-L-glutamyl desB30 insulin 101 N^(εB29)-10-(4-Carboxyphenoxy)decanoyl beta-Asp desB30 insulin 42 N^(εB29)-11-(4-Carboxy-phenoxy)undecanoyl-γ-L-glutamyl desB30 insulin 55

Example 28 Preparation of Monoclonal mIR Antibodies

Specific antibodies (F12) were produced by monoclonal technique: RBFmice were immunized by injecting 50 μg of purified mIR in FCAsubcutaneously followed by two injections with 20 μg of mIR in FIA.Highresponder mice were boosted intravenously with 25 μg of mIR and thespleens were harvested after 3 days. Spleen cells were fused with themyeloma Fox cell line (Köhler, G & Milstein C. (1976), European J.Immunology, 6:511-19; Taggart R T et al (1983), Science 219:1228-30).Supernatants were screened for antibody production in a mIR specificELISA. Positive wells were cloned and tested in Western blotting.

Example 29 Hydrophobicity Data on Insulin Derivatives According to theInvention

The hydrophobicity (hydrophobic index) of the insulin derivatives of theinvention relative to human insulin, k′_(rel), were measured on aLiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40°C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3,containing 10% acetonitrile, and B) 50% acetonitrile in water aseluents. The elution was monitored by following the UV absorption of theeluate at 214 nm. Void time, t₀, was found by injecting 0.1 mM sodiumnitrate. Retention time for human insulin, t_(human), was adjusted to atleast 2t₀ by varying the ratio between the A and B solutions.k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀). k′_(rel). found for anumber of insulin derivatives according to the invention.

Data on receptor binding and hydrophobicity data of insulin derivativesaccording to the present invention are shown in the following tables:Insulin derivative k′_(rel)N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB301.19 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₃CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB300.81 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-para-C₆H₄CO] desB30 human1.08 insulin N^(εB29)-[N—(HOOC(CH₂)₁₄CO)-γ-Glu-N-CH₂-para-C₆H₄CO] desB30human 1.12 insulinN^(εB29)-(3-Carboxy-5-hexadecandioylamino-benzoyl)desB30 insulin 1.7N^(εB29)-[N—(HOOC(CH₂)₁₅CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB301.23 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-CH₂-para-C₆H₄CO] desB302.03 human insulinN^(εB29)-[(5-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}furan-2-1.19 carbonyl)desB30 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-meta C₆H₄CO] desB301.63 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂-ortho C₆H₄CO] desB300.905 human insulinN^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-para-C₆H₄CO] desB30 human1.08 insulin N^(εB29)-(3-Carboxy-5-octadecandioylamino-benzoyl) des(B30)human insulin 2.97N^(εB29)-(3-Carboxy-4-(14-carboxy-tetradecyloxy)-benzoyl) desB30 humaninsulin 1.51 N^(εB29)-(3-Carboxy-5-(14-carboxy-tetradecyloxy)-benzoyl)desB30 human insulin 1.175N^(εB29)-{4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB300.388 human insulinN^(εB29)-[3-Carboxy-5-(octadecandioyl-N-carboxyethyl-glycin)amino-benzoyl]0.662 desB30 human insulinN^(εB29){3-[(3,5-Bis-carboxymethoxy-benzyl)-(15-carboxy-pentadecanoyl)-amino]-0.45 propionyl desB30 human insulinN^(εB29)-3-[4′-(2-Carboxy-ethyl)-biphenyl-4-yl]-propionyl-γ-L-glutamyldesB30 0.276 insulinN^(εB29)-hexadecandioyl-(4-aminomethyl-benzoyl)-γ-L-glutamyl desB30human 1.18 insulinN^(εB29)-4-{[(2-Carboxyethyl)-(15-carboxypentadecanoyl)amino]methyl}benzoyl)--0.399 γ-L-glutamyl desB30 human insulinN^(εB29)-{4-[2-(4-carboxymethyl-phenyl)-ethyl]-phenyl}-acetyl-γ-L-glutamyldesB30 0.291 human insulinN^(εB29)-(3-carboxy-4-hexdecandioylamino-benzoyl) desB30- insulin 1.075N^(εB29)-10-(4-carboxy-phenylsulfanyl) decanoyl--γ-L-glutamyl desB30human 0.475 insulin N^(εB29)--10-(4-Carboxyphenoxy) decanoyl beta-AspdesB30 insulin 0.348 N^(εB29)-11-(4-Carboxy-phenoxy)undecanoyl--γ-L-glutamyl desB30 human 0.482 insulin

1. An insulin derivative, said insulin derivative comprising a sidechain attached either to the α-amino group of the N-terminal amino acidresidue of the B chain or to an ε-amino group of a Lys residue presentin the A or the B chain of the parent insulin moiety via an amid bond,which side chain comprises at least one aromatic group; at least onefree carboxylic acid group or a group which is negatively charged atneutral pH, a fatty acid moiety with from 4 to 22 carbon atoms in thecarbon chain; and possible one or more linkers linking the individualcomponents in the side chain together via amide bonds, provided that thefatty acid moiety is not a divalent hydrocarbon chain of the formula—(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w are integers or one of themis zero so that the sum of v₄ and w₁ is in the range of 6 to
 30. 2. Theinsulin derivative according to claim 1, wherein the aromatic group isarylene or heteroarylene group which may be substituted with one or twogroups selected from —COOH, —SO₃H, —PO₃H₂ and tetrazolyl.
 3. The insulinderivative according to claim 2, wherein the heteroarylene groupcomprises nitrogen, oxygen or sulphur.
 4. The insulin derivativeaccording to claim 1, wherein the linker comprises 1-4 amino acidresidues linked together via amide bonds of which at least one has afree carboxylic acid group or a group which is negatively charged atneutral pH.
 5. The insulin derivative according to claim 1, wherein sidechain is attached to the ε-amino group of a Lys residue present in the Bchain of the parent insulin molecule.
 6. The insulin derivativeaccording to claim 5, wherein side chain is attached to the ε-aminogroup of LysB29.
 7. The insulin derivative according to claim 1 havingthe formula

wherein Ins is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofa Lys residue present in the B chain of the insulin moiety is bound tothe CO— group in the side chain via an amide bond; X₁ is selected fromthe group consisting of: —(CH₂)_(n) where n is 1, 2, 3, 4, 5 or 6; NR,where R is hydrogen or —(CH₂)_(p)—COOH; —(CH₂)_(p)SO₃H;—(CH₂)_(p)—PO₃H₂—(CH₂)_(p)—O—SO₃H₂; —(CH₂)_(p)—O—PO₃H₂; arylenesubstituted with 1 or 2 —(CH₂)_(p)—O—COOH groups; —(CH₂)_(p)-tetrazolyl,where p is an integer in the range of 1 to 6; —(CR₁R₂)_(q)—NR—CO—, whereR₁ and R₂ independently of each other and independently for each valueof q can be H, —COOH, or OH, q is 1-6 and R is defined as above;—((CR₃R₄)_(q1)—NR—CO)₂₋₄—, where R₃ and R₂ independently of each otherand independently for each value of q₁ can be H, —COOH, or OH, q₁ is 1-6and R is defined as above; and a bond, W is arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —COOH, —SO₃H, and —PO₃H₂ and tetrazolyl, or W is a bond; mis 0, 1, 2, 3, 4, 5 or 6; X is selected from the group consisting of: a)—O—;

where R is defined as above; or is a bond; Y is —(CR₁R₂)_(q)—NR—CO—,where R₁ and R₂ independently of each other and independently for eachvalue of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined asabove; NR where R is defined as above; —((CR₃R₄)_(q1)—NR—CO)₂₋₄—, whereR₃ and R₂ independently of each other and independently for each valueof q₁ can be H, —COOH, or OH, q₁ is 1-6 and R is defined as above; or abond, Q is —(CH₂)_(r)— where r is an integer from 4 to 22; a divalenthydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of—CH₂— groups sufficient to give a total number of carbon atoms in thechain in the range of 4 to 22; or a divalent hydrocarbon chain of theformula—(CH₂)_(s)-Q₁-(C₆H₄)_(v1)-Q₂-(CH₂)_(W)-Q₃-(C₆H₄)_(v2)-Q₄-(CH₂)_(t)-Q₅-(C₆H₄)_(v3)-Q₆-(CH₂)_(z)—wherein Q₁-Q₆ independently of each other can be O; S or a bond; wheres, w, t and z independently of each other are zero or an integer from 1to 10 so that the sum of s, w, t and z is in the range from 4 to 22, andv₁, v₂, and v₃ independently of each other can be zero or 1, providedthat when W is a bond then Q is not a divalent hydrocarbon chain of theformula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v₄ and w₁ are integers or oneof them is zero so that the sum of v₄ and w₁ is in the range of 6 to 22;and Z is selected from the group consisting of: —COOH; —CO-Asp; —CO-Glu;—CO-Gly; —CO-Sar; —CH(COOH)₂; —N(CH₂COOH)₂; —SO₃H; —PO₃H₂; O—SO₃H;O—PO₃H₂; -tetrazolyl and —O—W₁, where W₁ is arylene or heteroarylenesubstituted with one or two groups selected from —COOH, —SO₃H, and—PO₃H₂ and tetrazolyl; provided that if W is a bond and v₁, v₂ and v₃are all zero and Q₁₋₆ are all a bond, then Z is O—W₁ and any Zn²⁺complex thereof.
 8. The insulin derivative according to claim 7, whereinW is phenylene.
 9. The insulin derivative according to claim 7, whereinW is 5-7 membered heterocyclic ring system comprising nitrogen, oxygenor sulphur.
 10. The insulin derivative according to claim 9, wherein Wis a 5 membered heterocyclic ring system comprising at least one oxygen.11. The insulin derivative according claim 7, wherein Q is —(CH₂)_(r)—where r is an integer in the range of from 4 to 22, from 8- to 20, from12 to 20 or from 14-18.
 12. The insulin derivative according claim 7,wherein Q₁, Q₂, Q₅ and Q₆ are all a bond, v₂ is 1 and v₁ and v₃ arezero.
 13. The insulin derivative according claim 12, wherein Q₃ and Q₄are oxygen.
 14. The insulin derivative according claim 7, wherein X₁ andY are a bond and X is

where R is —(CH₂)_(p)—COOH, where p is 1-4.
 15. The insulin derivativeaccording to claim 7, wherein Z is —COOH.
 16. The insulin derivativeaccording to claim 1, wherein the parent insulin moiety is a des(B30)human insulin or an analogue thereof.
 17. The insulin derivativeaccording to claim 1, wherein the parent insulin moiety is selected fromthe group consisting of human insulin; des(B1) human insulin; desB30human insulin; GlyA21 human insulin; GlyA21des(B30)human insulin; AspB28human insulin; porcine insulin; LysB28ProB29 human insulin;GlyA21ArgB31ArgB32 human insulin; and LysB3GluB29 human insulin.
 18. Theinsulin derivative according to claim 1 selected from the groupconsisting of 0100-0000-0496N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂—C₆H₄CO] des(B30) humaninsulin; 0100-0000-0515N^(εB29)-[N—(HOOC(CH₂)₁₃CO)—N-(carboxyethyl)-CH₂—C₆H₄CO] des(B30) humaninsulin; 0100-0000-0522N^(εB29)-[N—(HOOC(CH₂)₁₅CO)—N-(carboxyethyl)-CH₂—C₆H₄CO] des(B30) humaninsulin; 0100-0000-0488N^(εB29)-[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-CH₂—C₆H₄CO] des(B30) humaninsulin; 0100-0000-0544N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-C₆H₄CO] des(B30) humaninsulin, and 0100-0000-029N^(εB29)-[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-CH₂— (furanylene)CO]des(B30) human insulin, 0100-0000-0552N^(εB29)-{4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB30 human insulin
 19. A pharmaceutical composition for the treatmentof diabetes in a patient in need of such treatment, comprising atherapeutically effective amount of an insulin derivative, said insulinderivative comprising a side chain attached either to the α-amino groupof the N-terminal amino acid residue of the B chain or to an ε-aminogroup of a Lys residue present in the A or the B chain of the parentinsulin moiety via an amid bond, which side chain comprises at least onearomatic group; at least one free carboxylic acid group or a group whichis negatively charged at neutral pH, a fatty acid moiety with from 4 to22 carbon atoms in the carbon chain; and possible one or more linkerslinking the individual components in the side chain together via amidebonds, provided that the fatty acid moiety is not a divalent hydrocarbonchain of the formula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w areintegers or one of them is zero so that the sum of v₄ and w₁ is in therange of 6 to 30 together with a pharmaceutically acceptable carrier.20. A pharmaceutical composition for the treatment of diabetes in apatient in need of such treatment, comprising a therapeuticallyeffective amount of an insulin derivative, said insulin derivativecomprising a side chain attached either to the α-amino group of theN-terminal amino acid residue of the B chain or to an ε-amino group of aLys residue present in the A or the B chain of the parent insulin moietyvia an amid bond, which side chain comprises at least one aromaticgroup; at least one free carboxylic acid group or a group which isnegatively charged at neutral pH, a fatty acid moiety with from 4 to 22carbon atoms in the carbon chain; and possible one or more linkerslinking the individual components in the side chain together via amidebonds, provided that the fatty acid moiety is not a divalent hydrocarbonchain of the formula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w areintegers or one of them is zero so that the sum of v₄ and w₁ is in therange of 6 to 30 in mixture with an insulin or an insulin analogue whichhas a rapid onset of action, together with a pharmaceutically acceptablecarrier.
 21. A method of treating diabetes in a patient in need of sucha treatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative, said insulin derivativecomprising a side chain attached either to the α-amino group of theN-terminal amino acid residue of the B chain or to an ε-amino group of aLys residue present in the A or the B chain of the parent insulin moietyvia an amid bond, which side chain comprises at least one aromaticgroup; at least one free carboxylic acid group or a group which isnegatively charged at neutral pH, a fatty acid moiety with from 4 to 22carbon atoms in the carbon chain; and possible one or more linkerslinking the individual components in the side chain together via amidebonds, provided that the fatty acid moiety is not a divalent hydrocarbonchain of the formula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w areintegers or one of them is zero so that the sum of v₄ and w₁ is in therange of 6 to 30 together with a pharmaceutically acceptable carrier.22. A method of treating diabetes in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative, said insulin derivativecomprising a side chain attached either to the α-amino group of theN-terminal amino acid residue of the B chain or to an ε-amino group of aLys residue present in the A or the B chain of the parent insulin moietyvia an amid bond, which side chain comprises at least one aromaticgroup; at least one free carboxylic acid group or a group which isnegatively charged at neutral pH, a fatty acid moiety with from 4 to 22carbon atoms in the carbon chain; and possible one or more linkerslinking the individual components in the side chain together via amidebonds, provided that the fatty acid moiety is not a divalent hydrocarbonchain of the formula —(CH₂)_(v4)C₆H₄(CH₂)_(W1)— wherein v and w areintegers or one of them is zero so that the sum of v₄ and w₁ is in therange of 6 to 30 in mixture with an insulin or an insulin analogue whichhas a rapid onset of action, together with a pharmaceutically acceptablecarrier.
 23. (canceled)